KR20100127842A - Vesicular targeting liposomes - Google Patents

Abstract

A composition is provided that includes a lipid particle, such as a liposome, that can fuse with an ER membrane of a cell. Lipid particles can also deliver contents encapsulated within the particles, such as therapeutic or imaging agents, into the ER lumen of the cell. The compositions may be useful for the treatment and / or prevention of diseases or conditions caused by or associated with viruses, such as viral infections such as HIV and HCV infections.

Description

Endoplasmic reticulum targeting liposomes {ENDOPLASMIC RETICULUM TARGETING LIPOSOMES}

<Related application>

This application claims priority to US Provisional Application No. 61 / 039,638, filed March 26, 2008, which application is incorporated herein by reference in its entirety.

<Technical Field>

The present application generally relates to methods and compositions for delivering active agents, such as therapeutic and / or imaging agents, and more particularly to methods and compositions for delivering active agents using lipid particles, such as liposomes.

One embodiment provides a method of drug delivery comprising administering to a host in need of drug delivery a composition comprising a lipid particle comprising one or more PS lipids.

Another embodiment comprises administering to a host in need of treatment or prevention of an HIV infection a composition comprising a lipid particle comprising at least one of PS lipids or PI lipids and free of CHEMS lipids. Or provide preventive measures.

Another embodiment provides a method of drug delivery comprising administering to a subject in need thereof a composition comprising a lipid particle comprising one or more polyunsaturated lipids.

Another embodiment provides a composition comprising a lipid particle comprising a PS lipid.

Another embodiment provides a composition comprising a lipid particle comprising one or more polyunsaturated lipids.

Another embodiment includes contacting a cell infected with a virus with a lipid particle comprising a) at least one of a PI or PS lipid and b) at least one labeled lipid comprising at least one label, wherein said contacting comprises the virus Labeling the virus, which results in labeling with the label.

1 (A) -1 (G) show the chemical structures of the following lipids: (A) DOPE; (B) DOPC; (C) CHEMS; (D) PI; (E) PS; (F) Rho-PE and (G) b-PE.
2 (A) -2 (H) show confocal microscopy images studying co-localization of the following liposomes with ER membrane protein EDEM in Huh7.5 cells: (A) PE: CH (molar ratio 3: 2) Liposomes; (B) PE: PC (3: 2) liposomes; (C) PE: CH: PI (3: 1: 1) liposomes; (D) PE: PC: PI (2: 2: 1) liposomes; (E) PE: CH: PS (3: 1: 1) liposomes; (F) PE: PC: PS (2: 2: 1) liposomes; (G) PE: CH: PI: PS (3: 1: 0.5: 0.5) liposomes; (H) PE: PC: PI: PS (1.5: 1.5: 1: 1) liposomes.
3 shows the calculated co-localization of EDEM antibodies with liposome-delivered rh-DOPE.
4 shows the percentage of tagged viral particles captured by streptavidin relative to the total amount of virion secreted (100%) in the same sample as a mole percentage function of b-PE in liposomes.
5 shows Rh-PE-tagged JC after incubation with uninfected Huh7.5 cells for 1 hour (MOI = 0.1) followed by washing the cells and incubating for additional 0, 6 or 24 hours in fresh medium. Confocal microscopy images of -1 HCVcc (red, bottom-left panel) are shown. After each incubation time, cells were fixed, stained with anti-HCV core antibody (green, top-right panel) and DAPI (blue, top-left panel), mounted on microscope slides and confocal microscopy images. The merged image is displayed in the bottom-right panel. Representative images from each incubation period are displayed. The resolution bar for each image is 10 μm.
6 (A) -6 (C) show PE: CH liposomes (A) at a molar ratio of 3: 2; Fluorescence microscopy images are shown to study the incorporation of PE: CH: PI (3: 1: 1) liposomes (B) and PE: CH: PS (3: 1: 1) liposomes (C) into cell membranes.
7 shows a plot showing increased intracellular influx and lipid retention of ER-targeted liposomes inside Huh7.5 cells. The data represent the mean and standard deviation (SD) of three samples from three independent experiments. The graph shows two sets of data: cell growth (dashed line) and rh-PE-liposomal influx (solid line) for both ER liposomes (black lines) and pH-sensitive liposomes (red lines). The Y-axis represents the maximum value for two sets of data normalized to 100%. The maximum value of 100% cell growth is 2.4x10e6 cells / ml (72 hours read in ER liposomes) and the maximum for rh-PE fluorescence is 1.5x10e-3 arbitrary units (AU) / cell (96 hours read in ER liposomes). )to be.
8 (A) is released from liposomes for maximum fluorescence determined by the addition of Triton X-100 to disrupt liposome membranes at the end of the incubation period as a function of time for PE: CH and PE: PC: PI: PS liposomes. Plot showing percentage of calcein taken.
8 (B) shows 10% bovine serum (FBS); Experimental results are shown for Rh-labeled liposomes (50 μM lipid concentration) incubated with Huh7.5 cells in the presence of 10% human serum or in serum-free medium for 24 hours. After incubation time, cells were harvested, counted and fluorescence measured at λex = 550 nm and λem = 590 nm. The results are expressed as the average fluorescence measured per cell for each sample. All data represent mean and SD of three samples from three independent experiments.
9 shows viability of Huh7.5 cells after 5 days incubation with different liposome formulations encapsulating 1 × PBS. Final lipid concentrations in the medium ranged from 0 to 500 μM. The results represent the mean value of three samples from three independent experiments.
10 shows the viability of PBMCs after 5 days incubation with different liposome formulations encapsulating 1 × PBS. Final lipid concentrations in the medium ranged from 0 to 500 μM. The results represent the mean value of three samples from three independent experiments.
11 shows secretion of HIV from infected PBMCs during treatment with liposomes for 5 days.
12 shows the infectivity of HIV virions secreted from liposome-treated HIV-infected PBMCs.
FIG. 13 shows experimental results for self-quenching calcein-loaded rh-PE-labeled liposomes (final lipid concentration of 50 μM) incubated with Huh7.5 cells in complete DMEM / 10% FBS for 45 minutes. . Intracellular dequenching of calcein from liposomes after incubation was measured at λex = 490 nm, λem = 520 nm, and total liposome influx was determined by fluorescence measurements at λex = 550 nm, λem = 590 nm during the same incubation period. . Assays were performed at 37 ° C. and 4 ° C. and all 4 ° C. values were subtracted from the 37 ° C. values to correct for liposome binding without endocytosis. By calculating the ratio of calcein dequenching and rh-PE fluorescence in treated cells after incubation, the ability of liposomes to deliver encapsulated calcein into Huh7.5 cells was measured. The data represent the mean and SD of three samples from three independent experiments.
FIG. 14 shows secretion of HIV from free PBMC: free versus liposome-mediated delivery during 5 days treatment with 1 mM NB-DNJ.
15 shows the infectivity of HIV virions secreted from NB-DNJ-liposomes or free NB-DNJ-treated HIV-infected PBMCs.
16 shows the viability of PBMCs after 5 days incubation with different liposome formulations encapsulating 1 mM NB-DNJ.
17 shows secretion of HCV from acutely and chronically-infected Huh7.5 cells after treatment with liposomes for 5 days.
18 shows the infectivity of HCV virions secreted from liposome-treated HCV-infected Huh7.5 cells, both acutely and chronically infected.
Figure 19 shows untreated Huh7.5 cells (left panel) and PE: PC: PI: PS liposome-treated Huh7.5 cells probed with BODIPY 493/503 (green) to visualize LD after 16 hour incubation. Confocal microscopy image of.
20 shows confocal microscopy images of Huh7.5 cells (left panel) treated with PE: PC: PI: PS liposomes for 2 hours and probed with LD stain (green). PE: PC: PI: PS liposomes were added to the cell culture medium at a final lipid concentration of 50 μM. DAPI (blue) was used as nuclear stain. The bottom-right panel is an integrated image. Yellow identifies areas of co-localization within the cell.
21A shows untreated Huh7.5 cells (left panel) and PE: PC: PI: PS liposome-treated Huh7 incubated for 16 hours and probed with anti-HCV core antibody (red) and LD stain (green) .5 shows confocal microscopy images of cells (right panel). FIG. 21B shows a close-up of an integrated image (white box in FIG. 9A) for untreated (left) and PE: PC: PI: PS (right) cells. 21C is a schematic of HCV core protein / LD interactions in the presence (right) and absence (left) of PE: PC: PI: PS liposomes.
22A-22D show the chemical structures of exemplary polyunsaturated lipids: 22: 6 PE (A); 20: 4 PE (B); 22: 6 PC (C) and 20: 4 PC (D).
23A to 23B respectively show the following. (23A) JC-1 HCVcc secretion from infected Huh7.5 cells (MOI = 0.5) during 4 day incubation in the presence of various ER liposome formulations was quantified from 500 μl of cell supernatant. Secretion is measured by quantification of JC-1 HCVcc RNA in the supernatant by quantitative PCR. (23B) Infectivity of secreted JC-1 HCVcc from liposome-treated JC-1-infected Huh7.5 cells. Infected with uninfected Huh7.5 cells for 1 hour, then incubated for 48 hours and fixed cells at this point, stained with anti-HCV core antibody to quantify the number of infected cells, and visualized all cells. Staining with DAPI determined the infectivity of secreted HCVcc.

Definition of term

Unless otherwise specified, the singular "a" or "an" means "one or more".

As used herein, the term “viral infection” refers to the virus invading healthy cells, to proliferating or replicating using the cell's reproductive apparatus, and ultimately to lysing the cells, resulting in cell death, release of viral particles and newly produced progeny virus. Refers to a diseased condition that results in the infection of other cells by. Latent infection by certain viruses is also a possible consequence of viral infection.

As used herein, the term “treatment or prevention of a viral infection” refers to the inhibition of replication of a particular virus, the inhibition of viral transmission, or preventing the virus from establishing itself in its host, and of the disease caused by the viral infection. It may mean alleviation or alleviation of symptoms. Treatment can be considered curative if it results in a reduction in viral load, a lethality rate and / or a reduction in morbidity.

The term “therapeutic agent” refers to an agent, such as a molecule or compound, that can help treat a physiological condition, such as a viral infection or a disease caused by it.

The term "liposome" may be defined as a particle comprising a lipid in the form of a bilayer, typically in the form of a spherical bilayer. Liposomes discussed herein can include one or more lipids represented by the following abbreviations:

CHEMS means cholesteryl hemisuccinate lipid.

DOPE means dioleoylphosphatidylethanolamine lipid.

DOPC means dioleoylphosphatidylcholine lipid.

PE means phosphatidylethanolamine lipid or a derivative thereof.

PEG-PE means PE lipid conjugated with polyethylene glycol (PEG). One example of PEG-PE may be polyethylene glycol-distearoylphosphatidylethanolamine lipid. The molecular weight of the PEG component of PEG can vary.

Rh-PE means lisamine rhodamine B-phosphatidylethanolamine lipid.

MCC-PE means 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [4- (p-maleimidomethyl) cyclohexane-carboxamide] lipid.

PI means phosphatidylinositol lipid.

PS means phosphatidylserine lipid.

The term “intracellular delivery” may refer to the delivery of encapsulated material from liposomes to any intracellular compartment.

IC50 or IC90 (inhibition concentration 50 or 90) may refer to the concentration of therapeutic agent used to achieve a 50% or 90% reduction in viral infection, respectively.

PBMC refers to peripheral blood mononuclear cells.

sCD4 refers to soluble CD4 molecules. "Soluble CD4" or "sCD4" or "D1D2" is a CD4 molecule that is in an aqueous solution and can mimic the activity of uninfected membrane-anchored CD4 by changes in the placement of HIV Env, as understood by one of ordinary skill in the art. Or a fragment thereof. One example of soluble CD4 is 2-domain soluble CD4 (sCD4 or D1D2), which is described, for example, in Salzwedel et al. J. Virol. 74: 326 333, 2000.

MAb means monoclonal antibody.

DNJ stands for deoxynojirimycin.

NB-DNJ stands for N-butyl deoxynojirimycin.

NN-DNJ represents N-nonyl deoxynojirimycin.

BVDV means bovine viral diarrhea virus.

HBV means hepatitis B virus.

HCV means hepatitis C virus.

HIV means human immunodeficiency virus.

Ncp means non-cell degeneration.

Cp means cell degeneration.

ER means endoplasmic reticulum.

CHO means Chinese hamster ovary cells.

MDBK means Madin-Darby bovine kidney cells.

PCR means polymerase chain reaction.

FOS means free oligosaccharides.

HPLC means high performance liquid chromatography.

PHA means phytohemagglutinin.

FBS means fetal bovine serum.

TCID50 refers to 50% tissue culture infection.

ELISA means enzyme-linked immunosorbent assay.

IgG means immunoglobulin.

DAPI means 4 ', 6-diamidino-2-phenylindole.

PBS means phosphate buffered saline.

LD stands for lipid droplet.

NS means unstructured.

"MOI" refers to multiplicity of infection.

<Related application>

This disclosure introduces US Patent Application Publication No. 2008-0138351 to its entirety.

Hepatitis C

Approximately 170 million people worldwide, or 3% of the world's population (see, for example, WHO, J. Viral. Hepat. 1999; 6: 35-47) and approximately 4 million people in the United States Is infected with (HCV, HepC). About 80% of individuals acutely infected with HCV become chronic infections. Therefore, HCV is a major cause of chronic hepatitis. When chronically infected, the virus is rarely removed without treatment. Rarely, HCV infection causes clinically acute diseases and even liver failure. Chronic HCV infection can vary dramatically between individuals, some people will have clinically meaningless or minimal liver disease, never develop complications, and others will have clinically evident chronic hepatitis, progression Can develop sclerosis. About 20% of individuals with HCV who develop sclerosis will develop terminal liver disease and have an increased risk of developing primary liver cancer.

Antiviral drugs (such as interferon alone or in combination with ribavirin) are effective in up to 80% of patients (Di Bisceglie, A. M, and Hoofnagle, JH 2002, Hepatology 36, S121-S127), but many patients Do not tolerate this form of combination therapy.

Geological droplet

Lipid droplets (LD) can be organelles that can be used for storage of neutral lipids. LD can move kinically through the cytoplasm and interact with other organelles, including ER. These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The HCV capsid protein (core) associates with the LD of the cell and can actively recruit nonstructural (NS) proteins and replication complexes to the LD-associated membrane for the production of infectious viral particles. HCV particles were observed in close proximity to the LD, indicating that several stages of viral assembly can occur around the LD (Miyanari et al, Nature Cell Biology, 9 (2007) pp. 1089-1097).

Human Immunodeficiency Virus (HIV)

HIV is the etiology of acquired immunodeficiency syndrome (AIDS) and related diseases. There are two or more completely different types of HIV: HIV-1 and HIV-2. In addition, there is a large amount of genetic heterogeneity within the populations of each of these types. Since AIDS has been epidemic, approximately 20 million people have died, more than 40 million live with HIV-1 / AIDS, and it is estimated that 14,000 are infected daily.

Numerous antiviral therapies and diagnostic capabilities have been developed, which have greatly improved the quality and quality of life, at least in approached persons. Most of these drugs interfere with viral proteins or processes such as reverse transcription and protease activity. Unfortunately, these treatments do not eliminate the infection, the unwanted effects of many therapies are severe, and drug resistant strains of HIV exist for all types of antiviral agents currently in use.

N-butyl-1,5-dideoxy-1,5-imino-D-glucitol

NB-DNJ (also known as N-butyl-1,5-dideoxy-1,5-imino-D-glucitol) can inhibit processing by ER glucosidase I and II, in particular To be an effective antiviral agent by causing incorrect folding and / or ER-retention of glycoproteins of HIV and hepatitis viruses such as hepatitis B virus (HBV), hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV) appear. Methods of synthesizing NB-DNJ and other N-substituted deoxynojirimycin derivatives are described, for example, in US Pat. Nos. 5,622,972, 4,246,345, 4,266,025, 4,405,714 and 4,806,650. The antiviral effects of NB-DNJ are described, for example, in US Pat. No. 6,465,487; No. 6,545,021; No. 6,689,759; 6,809,083 (effects on hepatitis virus) and US Pat. No. 4,849,430 (effects on HIV virus).

Glucosidase inhibitors such as NB-DNJ have been shown to be effective in the treatment of HBV infection in cell culture and Woodchuck animal models (see, eg, T. Block, X. Lu, AS Mehta, BS Blumberg, B. Tennant). , M. Ebling, B. Korba, DM Lansky, GS Jacob & RA Dwek, Nat Med. 1998 May; 4 (5): 610-4).

NB-DNJ inhibits secretion of HBV particles and induces intracellular retention of HBV DNA.

NB-DNJ has been shown to be a cell culture model for HCV, a strong antiviral agent for BVDV (see, for example, Branza-Nichita N, Durantel D, Carrouee-Durantel S, Dwek RA, Zitzmann N., J Virol. 2001 Apr; 75 (8): 3527-36; Durantel, D., et al, J. Virol, 2001, 75, 8987-8998; N. Zitzmann, et al, PNAS, 1999, 96, 11878 -11882). Treatment with NB-DNJ results in reduced infectivity of the virus offspring, which reduces the effect on the actual number of secreted viruses.

NB-DNJ has been shown to be an antiviral agent for HIV; Treatment results in a relatively small effect on the number of virus particles released from HIV-infected cells, but the amount of infectious virus released is greatly reduced (eg, PB Fischer, M. Collin, et al (1995). ), J. Virol 69 (9): 5791-7; PB Fischer, GB Karlsson, T. Butters, R. Dwek and F. Platt, J. Virol. 70 (1996a), pp. 7143-7152], (PB Fischer, GB Karlsson, R. Dwek and F. Platt, J. Virol. 70 (1996b), pp. 7153-7160). Clinical trials involving NB-DNJ were performed in HIV-1 infected patients, and the results demonstrated that the concentrations required for antiviral activity were too high and resulted in serious side effects in patients (see, eg, Fischl MA, Resnick L., Coombs R., Kremer AB, Pottage JC Jr, Fass RJ, Fife KH, Powderly WG, Collier AC, Aspinall RL, et.al., J. Acquir.Immune. Defic. Syndr. 1994 Feb; 7 2): 139-47). There is currently no mutant HIV strain resistant to NB-DNJ treatment.

ER protein folding and glucosidase I and II

The antiviral effect exhibited by glucosidase inhibition is believed to be the result of erroneous folding or retention of viral glycoproteins in the ER, primarily through blocking entry to the calnexin / caleticulin cycle. After transport of the triglucosylated oligosaccharide (Glc ₃ Man ₉ GlcNAc ₂ ) to the Asn-X-Ser / Thr consensus sequence in the growing polypeptide chain, three α may occur before further processing to mature carbohydrate units can occur. It is necessary for the bound glucose residues to be released. Moreover, two external glucose residues should be truncated to allow entry into the calnexin / caleticulin cycle for proper folding (see, eg, Bergeron, JJ et. Al., Trends Biochem. Sci., 1994). , 19, 124-128] (Peterson, JR et. Al., Mol. Biol. Cell, 1995, 6, 1173-1184). Initial processing is affected by ER-installed integral membrane enzymes (glucosidase I) with lumen-directed catalytic domains that specifically cleave α1-2 binding glucose residues; This is followed by the action of Glucosidase II, which releases both α1-3 binding glucose components.

Liposomes

Liposomes can deliver water-soluble compounds directly into cells by bypassing the cell membranes acting as molecular barriers. pH sensitive liposome formulations may comprise a combination of a compound containing an acidic group that acts as a stabilizer at neutral pH and phosphatidylethanolamine (PE) or a derivative thereof, such as DOPE. Cholesteryl hemisuccinate (CHEMS) may be a good stabilizing molecule because its cholesterol group confers higher stability to PE-containing vesicles compared to other amphoteric stabilizers in vivo. The in vivo efficacy of liposome-mediated delivery may be strongly dependent on the interaction with serum components (opsonine) that affect its pharmacokinetics and in vivo distribution. pH-sensitive liposomes can be rapidly cleared from the blood circulation and accumulate in the liver and spleen, but the inclusion of covalently attached polyethylene glycol (PEG) with lipids stabilizes the net-negative charge on DOPE: CHEMS liposomes. Overcoming cleaning by the system RES can result in long circulation times. DOPE-CHEMS and DOPE-CHEMS-PEG-PE liposomes and methods for their preparation are described, for example, in V.A. Slepushkin, S. Simoes, P. Dazin, M.S. Newman, L.S. Guo and M.C.P. de Lima, J. Biol. Chem. 272 (1997) 2382-2388; And [S. Simoes, V. Slepushkin, N. Duzgunes and M.C. Pedroso de Lima, Biomembranes 1515 (2001) 23-37, which is incorporated by reference in its entirety.

Delivery of NB-DNJ encapsulated in DOPE-CHEMS (molar ratio 6: 4) is disclosed in US patent application US2003 / 0124160.

Disclosure

We believe that lipid particles comprising one or more of PI or PS lipids (see FIG. 1), such as liposomes or micelles, can be efficiently taken up by and fused with the ER membranes of the cells. In addition, the inventors have found that lipid particles comprising at least one of PI or PS lipids may have high stability in blood or blood components such as serum. For example, liposomes comprising at least one of PI or PS lipids have higher stability in serum than DOPE / CHEMS liposomes (molar ratio 6: 3) or DOPE / CHEMS / PEG-PE (molar ratio 6: 3: 0.1) liposomes. Can have

In some embodiments, the lipid particles comprise at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or 3% to 60% or 5% to 50% of the PI and / or PS lipids. Or in a molar concentration of 10% to 30%. In some embodiments, the molar concentration of PS lipids in the lipid particles is at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or 3% to 60% or 5% to 50% or 10% to 30%. In some embodiments, the molar concentration of PI lipids in the lipid particles is at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or 3% to 60% or 5% to 50% or 10% to 30%. In some embodiments, the combined concentration of PI and PS lipids in the lipid particles is at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or 3% to 60% or 5% to 50. % Or 10% to 30%.

The lipid particle may further comprise one or more phosphatidylethanolamine (PE) lipids or derivatives thereof such as DOPE. In some embodiments, the PE lipids may comprise PE lipids conjugated with a label (eg, which may be a fluorophore label, biotin label or radiolabel). 1A, 1F and 1G show the chemical structures of DOPE lipids, Rho-PE lipids as an example of PE lipids conjugated with fluorophore labels, and b-PE lipids as an example of PE-lipids conjugated with biotin labels.

In some embodiments, the lipid particle may further comprise one or more of PC or CHEMS liposomes. In some other embodiments, the lipid particles may be free of PC and / or CHEMS lipids.

In some embodiments, lipid particles can be preferred, including PE, PC, PI, and PS lipids. Such lipid particles can interfere with the LD of the cells, which can result in a significant reduction in infectivity of HCV particles secreted from HCV-infected cells treated with these lipid particles. Lipid particles, including PE, PC, PI and PS lipids, can be used to introduce lipids into HCV-infected cells to interfere with LD / HCV core protein interactions. In addition, lipid particles, including PE, PC, PI and PS lipids, compete for the same cellular receptors as HCV, thus surpassing viral intracellular entry and reducing viral infectivity.

Virus infection

Lipid particles can be used for the treatment, prevention and / or monitoring of diseases or conditions caused by or associated with a virus in a subject (which in many cases may be a warm blooded animal such as a mammal or a bird). In many cases, the subject can be a human. In many cases, the disease or condition may be a viral infection.

In some embodiments, lipid particles comprising at least one of PI or PS lipids can be used for the treatment, prevention and / or monitoring of diseases or conditions caused by or associated with viruses belonging to the family Flaviviridae. have. Flaviviridae is a genus of Flaviviridae; Hepacivirus genus and pestivirus genus.

Flavivirus genera include Gadgets Gully virus (GGYV), Kadam virus (KADV); Kiyasana forest disease virus (KFDV); Langat virus (LGTV); Omsk hemorrhagic fever virus (OHFV); Powassan virus (POWV); Royal farm virus (RFV); Tick-borne encephalitis virus (TBEV); Leprosy virus (LIV); Meaban virus (MEAV); Samumarez Reef virus (SREV); Tyuleniy virus (TYUV); Aroa virus (AROAV); Dengue virus (DENV) 1-4; Kedougou virus (KEDV); Cascipacore virus (CPCV); Koutango virus (KOUV); Japanese encephalitis virus (JEV); Murray Valley Encephalitis Virus (MVEV); St. Louis Encephalitis Virus (SLEV); Usutu virus (USUV); Western Nile virus (WNV); Yaounde virus (YAOV); Kokobera virus (KOKV); Bagaza virus (BAGV); Ilheus virus (ILHV); Israel turkey meningoencephalitis virus (ITV); Ntaya virus (NTAV); Tembusu virus (TMUV); Zika virus (ZIKV); Banzi virus (BANV); Bouboui virus (BOUV); Edge Hill virus (EHV); Jugra virus (JUGV); Saboya virus (SABV); Sepik virus (SEPV); Uganda S virus (UGSV); Wesselsbron virus (WESSV); Yellow fever virus (YFV); Entebbe bat virus (ENTV); Yokose virus (YOKV); Apoi virus (APOIV); Cowbone Ridge virus (CRV); Jutiapa virus (JUTV); Modock virus (MODV); Sal Vieja virus (SVV); San Perlita Virus (SPV); Bukalasa bat virus (BBV); Carey Island Virus (CIV); Dakar bat virus (DBV); Montana myotis white encephalitis virus (MMLV); Phnom Penh bat virus (PPBV); Rio Bravo virus (RBV). The hepacivirus genus includes hepatitis C virus (HCV, Hep C). The pestivirus genus includes border disease viruses; Bovine diarrhea virus (BVDV) and typical swine fever virus. Diseases caused by or associated with flaviviruses include dengue fever; Japanese encephalitis; Kiyasana forest disease; Murray Valley Encephalitis; St. Louis Encephalitis; Tick-borne encephalitis; Western Nile encephalitis and yellow fever. Diseases caused by or associated with hepacivirus include hepatitis C virus infection. Diseases caused by or associated with pestiviruses include traditional swine fever (CSF) and bovine viral diarrhea (BVD) or bovine viral diarrhea / mucosal disease (BVD / MD).

In some embodiments, lipid particles can be used for the treatment, prevention and / or monitoring of diseases or conditions caused by or associated with viruses belonging to the family of Hepadnaviridae. The Hepadnaviridae family includes the genus Orthohepadnavirus, including hepatitis B virus, and the genus Avihepadnavirus, including duck hepatitis B virus. Diseases caused by or associated with hepadnavirus include hepatitis B virus infection.

In some embodiments, the lipid particles can be used for the treatment, prevention and / or monitoring of diseases or conditions caused by or associated with viruses belonging to the Retroviridae family. The retroviral family is a genus of alpharetroviruses, including the avian leukemia virus; Betaretrovirus genus, including mouse carcinoma virus; The genus Gamaretrovirus, including mouse leukemia virus and feline leukemia virus; The genus Deltaretrovirus, including bovine leukemia virus and human T-lymphophilic virus; Genus Epsilon retroviruses including corneal alum skin sarcoma virus; Lentivirus genera including human immunodeficiency virus 1, monkey immunodeficiency virus and feline immunodeficiency virus; The spumavirus genus, including chimpanzee foaming virus. Diseases and conditions caused by or associated with viruses belonging to the Retroviral family include HIV 1 infection.

In some embodiments, lipid particles may be used for the treatment, prevention and / or monitoring of diseases and conditions caused by or associated with glycoprotein containing viruses. Lipid particles can be used for the treatment and prevention of infections, such as viral infections, when administered as part of a composition to a subject such as a human. In some embodiments, such infection may be an infection caused by or associated with a glycoprotein containing virus, ie, a virus containing one or more glycoproteins. In some further embodiments, such infection may be a hepatitis infection, such as an HCV infection or an HBV infection. In some further embodiments, such infection may be a retroviral infection such as HIV. In some further embodiments, the infection may be a flavivirus infection, such as HCV.

If lipid particles are used for the treatment of HIV infection, this may reduce the infectivity of HIV particles secreted from HIV-infected cells.

If lipid particles are used for the treatment of HCV infection, this may interfere with the LD of the cell and reduce the infectivity of HCV particles secreted from HCV infected cells. In such cases lipid particles comprising PE, PC, PI and PS lipids may be preferred.

Although the present invention is not limited by its theory of operation, we believe that lipid particles comprising PE, PC, PI and PS lipids compete for the same cellular receptor as HCV, thus surpassing viral intracellular entry and reducing viral infectivity. I believe you can.

Active agent

In some embodiments, one or more agents, such as a therapeutic or imaging agent, can be encapsulated inside the lipid particle. Such agents can be, for example, water soluble molecules, peptides or amino acids. Compositions comprising lipid particles in combination with an encapsulated active agent can be used for the treatment, prevention or monitoring of diseases or conditions in which the active agent is known to be effective. The disease or condition may be any disease or condition in which intracellular delivery of the active agent may be beneficial.

The use of lipid particles containing PI and PS lipids can allow delivery of encapsulated material into the ER lumen of cells.

In some embodiments, the agent encapsulated inside the lipid particle can be an α-glucosidase inhibitor. In some embodiments, the α-glucosidase inhibitor may be an ER α-glucosidase inhibitor, which may be an ER α-glucosidase I inhibitor or an ER α-glucosidase II inhibitor. In general, any virus that depends on its interaction with calnexin and / or caleticulin for proper folding of its viral envelope glycoprotein can be targeted with an ER α-glucosidase inhibitor.

The alpha-glucosidase inhibitor is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, about 35, compared to the enzymatic activity of the alpha-glucosidase in the absence of an agent. An agent that inhibits host alpha-glucosidase enzymatic activity by at least%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or at least about 90% or more Can be. The term "alpha-glucosidase inhibitor" includes natural and synthetic agents that inhibit host alpha-glucosidase activity. Suitable alpha-glucosidase inhibitors include, but are not limited to, deoxynozirimycin and N-substituted deoxynojirimycins, such as the compounds of formula (I) and pharmaceutically acceptable salts thereof.

<Formula I>

Wherein R ₁ is a substituted or unsubstituted alkyl group which may be a branched or straight chain alkyl group; Substituted or unsubstituted cycloalkyl groups; Selected from substituted or unsubstituted aryl groups, substituted or unsubstituted oxaalkyl groups, substituted or unsubstituted arylalkyl, cycloalkylalkyl, and W, X, Y and Z are each independently hydrogen, alkanoyl Groups, aroyl groups and haloalkanoyl groups.

In some embodiments, R ₁ can be selected from a C1-C20 alkyl group or a C3-C12 alkyl group. In some embodiments, R ₁ is in ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, isopentyl, n-hexyl, heptyl, n-octyl, n-nonyl and n-decyl Can be selected. In some embodiments, R ₁ can be butyl or nonyl.

In some embodiments, R ₁ can be oxaalkyl, which can be a C1-C20 alkyl group or a C3-C12 alkyl group, and can also contain 1 to 5 or 1 to 3 or 1 to 2 oxygen atoms. have. Examples of oxaalkyl groups include-(CH ₂ ) ₂ O (CH ₂ ) ₅ CH ₃ ,-(CH ₂ ) ₂ O (CH ₂ ) ₆ CH ₃ ,-(CH ₂ ) ₆ OCH ₂ CH ₃ and-(CH ₂ ) ₂ OCH ₂ CH ₂ CH ₃ .

In some embodiments, R ₁ can be an arylalkyl group. Examples of arylalkyl groups include C1-C12-Ph groups such as C3-Ph, C4-Ph, C5-Ph, C6-Ph and C7-Ph.

In some embodiments, the compound of formula (I) is N- (n-hexyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (n-heptyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (n-octyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (n-octyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (n-decyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (n-undecyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (n-decyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (n-undecyl-)-1,5-dideoxy-1,5-imino-D-glucinol; N- (n-dodecyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (2-ethylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (4-ethylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (5-methylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (3-propylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (1-pentylpentylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (1-butylbutyl) -1,5-dideoxy-1,5-imino-D-glucinitol; N- (7-methyloctyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (8-methylnonyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (9-methyldecyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (10-methylundecyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (6-cyclohexylhexyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N- (4-cyclohexylbutyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (2-cyclohexylethyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (1-cyclohexylmethyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (1-phenylmethyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (3-phenylpropyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (3- (4-methyl) -phenylpropyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (6-phenylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol; N- (n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (n-decyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (n-undecyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (n-dodecyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (2-ethylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (4-ethylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (5-methylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (3-propylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (1-pentylpentylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (1-butylbutyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (7-methyloctyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (8-methylnonyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (9-methyldecyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (10-methylundecyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (6-cyclohexylhexyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (4-cyclohexylbutyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (2-cyclohexylethyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (1-cyclohexylmethyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (1-phenylmethyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (3-phenylpropyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (3- (4-methyl) -phenylpropyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; N- (6-phenylhexyl) -1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate; Pharmaceutically acceptable salts thereof; And mixtures of any two or more thereof.

Diseases and conditions in which N-substituted deoxynojirimycin may be effective are described in US Pat. No. 4,849,430; 4,876,268; 5,411,970; 5,411,970; 5,472,969; 5,472,969; No. 5,643,888; No. 6,225,325; No. 6,465,487; No. 6,465,488; No. 6,515,028; No. 6,689,759; 6,809,083; 6,809,083; 6,583,158; No. 6,589,964; 6,599,919; 6,599,919; No. 6,916,829; 7,141,582. Diseases and conditions in which N-substituted deoxynojirimycin may be effective include HIV infection; Hepatitis infections (including hepatitis C and hepatitis B infections); Lysosomal lipid accumulators such as Tay-Saxe, Gaucher's, Crabe's and Fabry's disease; And cystic fibrosis.

In some embodiments, the α-glucosidase inhibitor is selected from N-oxaalkylated deoxynojirimycin or N-alkyloxy deoxynojirimycin, such as N-hydroxyethyl DNJ (miglytol as described in US Pat. No. 4,639,436). (Miglitol) or Glyset®.

In some embodiments, the α-glucosidase inhibitor is a castanospermin and / or castanospermin derivative, such as a compound of Formula I as disclosed in US Patent Application 2006/0194835 and pharmaceutically acceptable salts thereof (6-O- Butanoyl castanospermine (including selgocivir) and the compounds of formula (II) disclosed in PCT Publication No. WO01054692 and pharmaceutically acceptable salts thereof.

Diseases and conditions in which castanospermine and derivatives thereof may be effective are described in US Pat. No. 4,792,558; 4,837,237; 4,837,237; 4,925,796; No. 4,952,585; 5,004,746; 5,214,050; 5,214,050; 5,264,356; 5,264,356; 5,385,911; 5,385,911; No. 5,643,888; 5,691,346; 5,691,346; 5,750,648; 5,750,648; 5,837,709; 5,837,709; 5,908,867; No. 6,136,820; No. 6,583,158; No. 6,589,964; 6,656,912 and US Publication No. 20020006909; No. 20020188011; US20020093577; 20060194835; No. 20080131398. Diseases and conditions in which castanospermine and its derivatives may be effective include retroviral infections such as HIV infection; Brain malaria; Hepatitis infections such as hepatitis B and hepatitis C infections; Diabetes and lysosomal accumulators include but are not limited to.

In some embodiments, the alpha glucosidase inhibitor is acarbose (O-4,6-dideoxy-4-[[(1S, 4R, 5S, 6S) -4,5,6-trihydroxy-3- (hydroxy) Oxymethyl) -2-cyclohexen-1-yl] amino] -α-D-glucopyranosyl- (1 → 4) -O- → -D-glucopyranosyl- (1 → 4) -D-glucose) Or Precos®. Acarbose is disclosed in US Pat. No. 4,904,769. In some embodiments, the alpha glucosidase inhibitor can be a highly purified form of acarbose (see, eg, US Pat. No. 4,904,769).

In some embodiments, the agent encapsulated inside the liposome can be an ion channel inhibitor. In some embodiments, the ion channel inhibitor may be an agent that inhibits the activity of HCV p7 protein. Ion channel inhibitors and methods of identifying them are described in detail in US Patent Publication No. 2004/0110795. Suitable ion channel inhibitors include compounds of formula I and their pharmaceutically acceptable salts, for example N- (7-oxa-nonyl) -1,5,6-trideoxy-1,5-imino-D-galac Titol (N-7-oxa-nonyl 6-MeDGJ or UT231B) and N-10-oxoundecyl-6-MeDGJ. Suitable ion channel inhibitors also include, but are not limited to, N-nonyl deoxynojirimycin, N-nonyl deoxynogalactonozirimycin, and N-oxanonyl deoxynogalactonozirimycin.

In some embodiments, the agent encapsulated inside the liposome can be an iminosugar. Suitable iminosugars include both natural iminosugars and synthetic iminosugars.

In some embodiments, the iminosugars can be deoxynojirimycin or N-substituted deoxynojirimycin derivatives. Examples of suitable N-substituted deoxynojirimycin derivatives include the compounds of formula (II) herein, the compounds of formula (I) in US Pat. No. 6,545,021 and the N-oxaalkylated deoxynojirimycin, such as N-described in US Pat. No. 4,639,436. Hydroxyethyl DNJ (miglitol or glycet®), including but not limited to.

In some embodiments, the iminosugars can be castanospermine or castanospermin derivatives. Suitable castanospermine derivatives include compounds of formula (I) and pharmaceutically acceptable salts thereof, as disclosed in US Patent Application 2006/0194835, and compounds of formula (II) and pharmaceutically acceptable salts thereof, as disclosed in PCT Publication No. WO01054692. This is not restrictive. In some embodiments, the iminosugars may be those disclosed in deoxynogalactosirimycin or N-substituted derivatives thereof, such as in PCT Publication Nos. WO99 / 24401 and WO01 / 10429. Examples of suitable N-substituted deoxynogalactozirimycin derivatives include N-alkylated deoxynogalactozirimycin (N-alkyl-1,5-dideoxy-1,5-imino-D-galactitol), such as N-nonyl deoxynogalactozirimycin, and N-oxa-alkylated deoxynogalactojirimycin (N-oxa-alkyl-1,5-dideoxy-1,5-imino-D-galactitol), Such as, but not limited to, N-7-oxanonyl deoxynogalactozirimycin.

In some embodiments, the iminosugars include, but are not limited to, N-substituted 1,5,6-trideoxy-1,5-imino-D-galactitol (N-substituted MeDGJ) Can be.

<Formula II>

Wherein R is selected from a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocyclyl group, or a substituted or unsubstituted oxaalkyl group. In some embodiments, substituted or unsubstituted alkyl groups and / or substituted or unsubstituted oxaalkyl groups comprise 1 to 16 carbon atoms, or 4 to 12 carbon atoms, or 8 to 10 carbon atoms. . In some embodiments, substituted or unsubstituted alkyl groups and / or substituted or unsubstituted oxaalkyl groups comprise 1 to 4 oxygen atoms, and in other embodiments 1 to 2 oxygen atoms. In other embodiments, substituted or unsubstituted alkyl groups and / or substituted or unsubstituted oxaalkyl groups comprise 1 to 16 carbon atoms and 1 to 4 oxygen atoms. Therefore, in some embodiments, R is-(CH ₂ ) ₆ OCH ₃ ,-(CH ₂ ) ₆ OCH ₂ CH ₃ ,-(CH ₂ ) ₆ O (CH ₂ ) ₂ CH ₃ ,-(CH ₂ ) ₆ O (CH ₂ ) ₃ CH ₃ ,-(CH ₂ ) ₂ O (CH ₂ ) ₅ CH ₃ ,-(CH ₂ ) ₂ O (CH ₂ ) ₆ CH ₃ and-(CH ₂ ) ₂ O (CH ₂ ) ₇ CH ₃ , but not limited thereto. N-substituted MeDGJ is disclosed, for example, in PCT Publication WO01 / 10429.

In some embodiments, the agent encapsulated inside the liposome may comprise a nitrogen containing compound of formula III or a pharmaceutically acceptable salt.

<Formula III>

Wherein R ¹² is alkyl such as C ₁ -C ₂₀ or C ₁ -C ₆ or C ₇ -C ₁₂ or C ₈ -C ₁₆ , and also 1 to 5 or 1 to 3 or 1 to 2 oxygen And R ¹² may be an oxa-substituted alkyl derivative. Examples of oxa-substituted alkyl derivatives include 3-oxanonyl, 3-oxadecyl, 7-oxanonyl and 7-oxadedecyl.

이며, 여기서 n은 3 또는 4이고, X는 각각 독립적으로 수소, 히드록시, 아미노, 카르복시, C₁-C₄ 알킬카르복시, C₁-C₄ 알킬, C₁-C₄ 알콕시, C₁-C₄ 히드록시알킬, C₁-C₆ 아실옥시 또는 아로일옥시이고, Y는 각각 독립적으로 수소, 히드록시, 아미노, 카르복시, C₁-C₄ 알킬카르복시, C₁-C₄ 알킬, C₁-C₄ 알콕시, C₁-C₄ 히드록시알킬, C₁-C₆ 아실옥시, 아로일옥시 또는 결실 (즉, 존재하지 않음)이고;R ² is hydrogen and R ³ is carboxy or C ₁ -C ₄ alkoxycarbonyl, or R ² and R ³ together

Wherein n is 3 or 4, and X is each independently hydrogen, hydroxy, amino, carboxy, C ₁ -C ₄ alkylcarboxy, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₆ acyloxy or aroyloxy, Y is each independently hydrogen, hydroxy, amino, carboxy, C ₁ -C ₄ alkylcarboxy, C ₁ -C ₄ alkyl, C _1- C ₄ alkoxy, C ₁ -C ₄ hydroxyalkyl, C ₁ -C ₆ acyloxy, aroyloxy or a deletion (ie not present);

R ⁴ is hydrogen or deleted (ie not present);

R ⁵ is hydrogen, hydroxy, amino, substituted amino, carboxy, alkoxycarbonyl, aminocarbonyl, alkyl, aryl, aralkyl, alkoxy, hydroxyalkyl, acyloxy or aroyloxy, or R ³ and R ⁵ together form phenyl and R ⁴ is deleted (ie not present).

In some embodiments, the nitrogen containing compound has the formula:

In the above formulas, R ⁶ to R ¹⁰ are each independently hydrogen, hydroxy, amino, carboxy, C ₁ -C ₄ alkylcarboxy, C ₁ -C ₄ alkyl, C ₁ -C ₄ alkoxy, C ₁ -C ₄ hydroxide Oxyalkyl, C ₁ -C ₄ acyloxy and aroyloxy; R ¹¹ is hydrogen or C ₁ -C ₆ alkyl.

Nitrogen-containing compounds include N-alkylated piperidine, N-oxa-alkylated piperidine, N-alkylated pyrrolidine, N-oxa-alkylated pyrrolidine, N-alkylated phenylamine, N- It may be oxa-alkylated phenylamine, N-alkylated pyridine, N-oxa-alkylated pyridine, N-alkylated pyrrole, N-oxa-alkylated pyrrole, N-alkylated amino acid or N-oxa-alkylated amino acid have. In certain embodiments, the N-alkylated piperidine, N-oxa-alkylated piperidine, N-alkylated pyrrolidine or N-oxa-alkylated pyrrolidine compound may be an iminosugar. For example, in some embodiments, the nitrogen-containing compound is N-alkyl-1,5-dideoxy-1,5-imino-D-galactitol (N-alkyl-DGJ) or N- having the formula Oxa-alkyl-1,5-dideoxy-1,5-imino-D-galactitol (N-oxa-alkyl-DGJ)

Or N-alkyl-1,5,6-trideoxy-1,5-imino-D-galactitol (N-alkyl-MeDGJ) or N-oxa-alkyl-1,5,6- Trideoxy-1,5-imino-D-galactitol (N-oxa-alkyl-MeDGJ):

Groups as used herein have the following characteristics unless the number of carbon atoms is specified otherwise. Alkyl groups have 1 to 20 carbon atoms and are linear or branched, substituted or unsubstituted. Alkoxy groups have 1 to 16 carbon atoms and are linear or branched, substituted or unsubstituted. Alkoxycarbonyl groups are ester groups having 2 to 16 carbon atoms. Alkenyloxy groups have 2 to 16 carbon atoms, 1 to 6 double bonds, and are linear or branched, substituted or unsubstituted. Alkynyloxy groups have 2 to 16 carbon atoms, 1 to 3 triple bonds, and are linear or branched, substituted or unsubstituted. Aryl groups have 6 to 14 carbon atoms (eg phenyl groups) and are substituted or unsubstituted. Aralkyloxy (eg benzyloxy) and aroyloxy (eg benzoyloxy) groups have 7 to 15 carbon atoms and are substituted or unsubstituted. The amino group can be a primary, secondary, tertiary or quaternary amino group (ie, substituted amino group). Aminocarbonyl groups are amido groups having 1 to 32 carbon atoms (eg, substituted amido groups).

Substituted groups can include a substituent selected from halogen, hydroxy, C _{1 -10} alkyl, C _{2 -10} alkenyl, C _{1 -10} the group consisting of acyl, or C _{1 -10} alkoxy.

N-alkylated amino acids may be N-alkylated natural amino acids such as N-alkylated a-amino acids. Natural amino acids include 20 common α-amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Asn, Lys, Glu, Gln, Arg, His, Phe, Cys, Trp, Tyr, Met and Pro) And other amino acids that are natural products such as norleucine, ethylglycine, ornithine, methylbutenyl-methylthreonine, and phenylglycine. Examples of amino acid side chains (eg, R ⁵ ) include H (glycine), methyl (alanine), -CH ₂ C (O) NH ₂ (asparagine), -CH ₂ -SH (cysteine), and -CH (OH) CH ₃ (threonine).

N-alkylated compounds can be prepared by reductive alkylation of amino (or imino) compounds. For example, an amino or imino compound can be exposed to an aldehyde with a reducing agent (eg sodium cyanoborohydride) to N-alkylate the amine. Similarly, N-oxa-alkylated compounds can be prepared by reductive alkylation of amino (or imino) compounds. For example, an amino or imino compound can be exposed to oxa-aldehyde with a reducing agent (eg, sodium cyanoborohydride) to amine N-oxa-alkylation.

The nitrogen-containing compound may comprise one or more protecting groups. Various protecting groups are well known. In general, the type of protecting group is not critical if it is stable to the conditions of any subsequent reaction (s) to other positions of the compound and does not deleteriously affect the rest of the molecule and can be removed at a suitable point in time. In addition, a protecting group may be substituted for another after substantial synthetic transformations have been completed. Obviously, the compounds are included in the present invention, provided that the compounds differ from the compounds disclosed herein only in that one or more protecting groups of the disclosed compounds are substituted with different protecting groups. Additional examples and conditions can be found in Greene, Protective Groups in Organic Chemistry, (1 ^st Ed., 1981, Greene & Wuts, 2 ^nd Ed., 1991).

Nitrogen-containing compounds can be purified, for example, by crystallization or chromatography methods. Compounds can be prepared stereospecifically using stereospecific amino or imino compounds as starting materials.

Amino and imino compounds used as starting materials in the preparation of long chain N-alkylated compounds are commercially available (Sigma, St. Louis, MO; Cambridge Research Biochemicals, Kesher Norwich, UK). Toronto Research Chemicals, Ontario, Canada, which may be prepared by known synthetic methods. For example, the compound may be an N-alkylated iminosugar compound or an oxa-substituted derivative thereof. Iminosugars are, for example, deoxygalactonozirimycin (DGJ), 1-methyl-deoxygalactonozirimycin (MeDGJ), deoxynojirimycin (DNJ), altrostatin, 2R, 5R- Dihydroxymethyl-3R, 4R-dihydroxypyrrolidine (DMDP), or derivatives thereof, enantiomers or stereoisomers.

In some embodiments, the agent encapsulated inside the lipid particle can be a compound of Formula IV or V below.

<Formula IV>

(V)

이고;In the above formulas, R is

ego;

이고;R 'is

ego;

R ₁ is a substituted or unsubstituted alkyl group; R ₂ is a substituted or unsubstituted alkyl group; W _{1 -4} is hydrogen, substituted or unsubstituted alkyl group, a substituted or unsubstituted haloalkyl group, with a substituted or unsubstituted alkanoyl group, a substituted or unsubstituted aroyl group, or a substituted or Independently selected from unsubstituted haloalkanoyl groups; X _{1 -5} are independently selected from H, NO _2, N ₃ or NH _2; Y is a substituted or unsubstituted C ₁ -alkyl group which is absent or is not carbonyl; Z is selected from a bond or NH; With the proviso that Y is absent when Z is a bond, and when Z is NH, Y is a substituted or unsubstituted C ₁ -alkyl group that is not carbonyl; Z 'is a bond or NH. Compounds of formulas IV and V and methods of their synthesis are disclosed, for example, in US Publication No. US2007 / 0275998. Non-limiting examples of compounds of Formulas IV and V include N- (N '-{4'-azido-2'-nitrophenyl) -6-aminohexyl) -deoxynojirimycin (NAP-DNJ) and N- (N '-{2,4-dinitrophenyl) -6-aminohexyl) -deoxynojirimycin (NDP-DNJ) is included.

The synthesis of various iminosugar compounds is described. For example, methods of synthesizing DNJ derivatives are known and are described, for example, in US Pat. Nos. 5,622,972, 5,401,645, 5,200,523, 5,043,273, 4,994,572, 4,246,345, 4,266,025, and 4,405,714. And 4,806,650. Methods of synthesizing other iminosugar derivatives are known and are described, for example, in U.S. Pat. 5,286,877 and 5,100,797, and PCT Publication WO 01/10429. Enantiomer specific synthesis of 2R, 5R-dihydroxymethyl-3R, 4R-dihydroxypyrrolidine (DMDP) is described in Fleet & Smith (Tetrahedron Lett. 26: 1469-1472, 1985). have.

Imaging agents can be tagged or fluorescent aqueous materials such as calcein, or fluorescently labeled molecules such as siRNA, antibodies, or other small molecule inhibitors. Tagged lipophilic materials can also be incorporated into lipid particles for incorporation into cell membranes, such as rh-PE lipids used for visualization of liposomes in cells, and other similar lipids with tags used for visualization or purification. Can be. It may also include a tagged lipophilic protein or fluorescent moiety or a drug with another tag for visualization or purification.

Targeting moiety

In some embodiments, a composition comprising lipid particles may comprise one or more targeting moieties, which may be conjugated with or inserted into the lipid layer or bilayer of the particle. In some embodiments, the targeting moiety can be a ligand that can be a ligand of the envelope protein of the virus, or an antibody that can be an antibody against the envelope protein of the virus. Such residues can be used to target the particles to cells infected with the virus. Such targeting moieties can also be used to achieve sterile immunity against viral infections associated with or caused by a virus.

In some embodiments, the targeting moiety can include a gp120 / gp41 targeting moiety. In such cases, compositions comprising lipid particles may be desirable for the treatment and / or prevention of HIV-1 infection. The gp120 / gp41 targeting moiety may comprise an sCD4 molecule or a monoclonal antibody such as an IgG 2F5 or IgG b12 antibody.

In some embodiments, the targeting moiety may comprise an El or E2 targeting moiety, such as an El or E2 protein from HCV. In such cases, compositions comprising lipid particles may be desirable for the treatment and / or prevention of HCV infection. In some cases, the targeting moiety may also be a molecule capable of targeting El and / or E2 proteins, such as specific antibodies to these proteins, and soluble portions of cellular receptors, such as soluble CD81 or SR-BI molecules.

Inserted residue

In some embodiments, a lipid particle may comprise one or more residues inserted into its lipid layer or bilayer. Examples of inserted residues include, but are not limited to, transmembrane proteins, protein lipid conjugates, labeled lipids, lipophilic compounds, or any combination thereof.

In some embodiments, the inserted moiety may comprise a lipid-PEG conjugate. Such conjugates can increase the in vivo stability of lipid particles and / or increase their circulation time.

In some embodiments, the inserted moiety is a long chain alkyl iminosugar, such as C7-C16 alkyl or oxaalkyl substituted N-deoxynojirimycin (DNJ) or C7-C16 alkyl or oxaalkyl substituted deoxygalactonoziri Mycin (DGJ). Non-limiting examples of long chain alkyl iminosugars include N-nonyl DNJ and N-nonyl DGJ.

In some embodiments, the inserted moiety can comprise a fluorophore-lipid conjugate that can be used to label the ER membrane of the cell in contact with the lipid bilayer particle. Such labeling may be useful for live and / or fixed-cell imaging in eukaryotic cells.

The use of lipid particles comprising PI and / or PS lipids can result in the delivery of inserted residues to the ER membrane of the cell.

Polyunsaturated lipid particles

We also believe that lipid particles, such as liposomes comprising one or more polyunsaturated lipids, may be effective in the treatment and / or prevention of infections, such as viral infections, in subjects such as humans.

In some embodiments, the polyunsaturated lipid is at least 5 mol% or at least 10 mol% or at least 15 mol% or at least 20 mol% or at least 25 mol% or at least 30 mol% or at least 35 mol% of the total lipids of the lipid particles or At least 40 mol% or at least 45 mol% or at least 50 mol% or at least 55 mol% or at least 60 mol% or at least 65 mol% or at least 70 mol% or at least 75 mol% or at least 80 mol% or at least 85 mol% or 90 mol% or more or 95 mol% or more can be comprised.

As used herein, the term "polyunsaturated lipid" refers to a lipid containing more than one unsaturated chemical bond, such as a double or triple bond, in its hydrophobic tail.

In some embodiments, the polyunsaturated lipid may have 2 to 8 or 3 to 7 or 4 to 6 double bonds in its hydrophobic tail.

As used herein, the term “polyunsaturated lipid particle” refers to a lipid particle comprising one or more polyunsaturated lipids.

In some embodiments, the lipid particle may comprise more than one polyunsaturated lipid.

Preferably, the polyunsaturated lipid particles contain at least one of polyunsaturated PE or polyunsaturated PC lipids. 22A-22D provide the chemical structures of exemplary polyunsaturated PE and PC lipids.

The lipid particle may further comprise one or more additional lipids, such as PI, PS or CHEMS.

Polyunsaturated lipid particles comprising at least one of polyunsaturated PE or polyunsaturated PC lipids can be used for the treatment, prevention, and monitoring of diseases or conditions caused or associated with viruses, such as the diseases or conditions disclosed above. In many embodiments, such disease or condition can be a viral infection. In some embodiments, such infection may be a hepatitis infection, such as an HCV infection or an HBV infection. In some further embodiments, such infection may be a retroviral infection such as HIV. In some further embodiments, the infection may be a flavivirus infection, such as an HCV infection.

In some embodiments, the polyunsaturated lipid particles may encapsulate one or more active agents, such as the agents disclosed above.

In some embodiments, the polyunsaturated lipid particle may comprise one or more residues inserted into the lipid layer or bilayer of the particle, which may be any inserted residues disclosed above.

In some embodiments, a composition comprising lipid particles may comprise a targeting moiety associated with the particle, which in turn may be any targeting moiety disclosed above.

In some embodiments, polyunsaturated lipid particles, including PE, PC, PI, and PS lipids, one or more of which are unsaturated, may be desirable for the treatment or prevention of HCV infection. The present invention is not limited by its theory of operation, but we believe that the delivery of polyunsaturated lipids to the ER membrane, the HCV replication site, can reduce HCV RNA replication and subsequently HCV secretion, resulting in PE, PC, PI and It is believed that polyunsaturated lipid particles, including PS lipids, can significantly reduce the secretion of HCV virions from HCV-infected cells.

administration

In some embodiments, a composition comprising lipid particles can be administered to a cell. The cell may be a cell infected with a virus. In many cases, the contacted cells can be cells from warm blooded animals, such as mammals or birds. In some embodiments, the contacted cells can be cells from humans.

In some embodiments, a composition comprising lipid particles can be administered to an individual. The subject may be a warm blooded animal such as a mammal or a bird. In many cases, the subject can be a human. In some embodiments, a composition comprising lipid particles can be administered by intravenous injection. In some further embodiments, compositions comprising lipid particles may be administered via parenteral routes other than intravenous injection, such as intraperitoneal, subcutaneous, intradermal, epidermal, intramuscular or transdermal routes. In some further embodiments, compositions comprising lipid particles may be administered through mucosal surfaces, such as ocular, intranasal, lung, intestinal, rectal and urinary tract surfaces.

Routes of administration for lipid containing compositions, such as liposome compositions, are described, for example, in A. S. Ulrich, Biophysical Aspects of Using Liposomes as Delivery Vehicles, Bioscience Reports, Volume 22, Issue 2, Apr 2002, 129-150.

Delivery of a therapeutic agent, such as NB-DNJ, to a ER lumen via lipid particles, such as liposomes, can lower the effective amount of therapeutic agent required for the inhibition of ER-glucosidase compared to non-liposomal methods. For example, for NB-DNJ, IC 90 may be reduced by at least 100, or by at least 500, or by at least 1000, or by at least 5000, or by at least 10000, or by at least 50000, or by at least 100,000. This reduction in the antiviral effective amount of NB-DNJ can result in a final concentration of NB-DNJ administered several tens of times lower than the toxicity level in mammals, especially humans.

In some cases, a composition comprising a lipid particle comprising a therapeutic agent, such as NB-DNJ, may be contacted with an infected cell in combination with one or more additional therapeutic agents, such as an antiviral agent. In some cases, such additional therapeutic agents may be co-encapsulated into lipid particles with NB-DNJ. In some additional cases, contact of such additional therapeutic agents with infected cells may be the result of administration of the additional therapeutic agent to a subject comprising the cells. Administration of the additional therapeutic agent can be carried out by adding the therapeutic agent to the composition. In some additional cases, administration of the additional therapeutic agent may be performed separately from administration of a composition comprising lipid particles containing NB-DNJ. Such separate administration may be carried out via a route of administration that may be the same or different than the route of administration used for the composition comprising the lipid particles.

Combination therapy can reduce the effective amount of the agent required for antiviral activity and thereby reduce its toxicity, as well as improve the absolute antiviral effect as a result of attacking the virus through multiple mechanisms.

Combination therapy may also provide a means to avoid or reduce the chance of developing viral resistance.

The particular additional therapeutic agent (s) that can be used in combination with liposomes containing NB-DNJ may depend on the disease or condition to be treated. For example, for hepatitis infections, such as HBV, HCV or BVDV infections, such therapeutic agent (s) may be nucleoside or nucleotide antiviral agents and / or immunostimulatory / immunomodulators. Various nucleoside agonists, nucleotide agonists and immunostimulatory / immunomodulators that can be used in combination with NB-DNJ for the treatment of hepatitis are described in US Pat. No. 6,689,759 (Jacob et. Al.), Issued February 10, 2004. Is illustrated. For example, for the treatment of hepatitis C infection, NB-DNJ is a 1-bD-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide (ribavirin) as nucleoside agonist, And in combination with interferon, such as interferon alpha, as an immunostimulant / immunomodulator. Treatment of hepatitis infection with ribavirin and / or interferon is described, for example, in US Pat. No. 6,172,046; No. 6,177,074; 6,299,872; 6,299,872; No. 6,387,365; 6,472,373; 6,472,373; 6,524,570 and 6,824,768.

For the treatment of HIV infection, the therapeutic agent that can be used in combination with liposomes containing NB-DNJ can be an anti-HIV agent, which is, for example, a nucleoside reverse transcriptase (RT) inhibitor such as (-)- 2'-deoxy-3'-thiocytidine-5'-triphosphate (3TC); (-)-Cis-5-fluoro-1- [2- (hydroxy-methyl)-[1,3-oxathiolan-5-yl] cytosine (FTC); 3'-azido-3'-deoxythymidine (AZT) and dideoxy-inosine (ddI); Non-nucleoside RT inhibitors such as N11-cyclopropyl-4-methyl-5,11-dihydro-6H-dipyrido [3,2-b: 2'3'-e]-[1,4] dia Zepin-6-one (nevirapine), a protease inhibitor, or a combination thereof. Anti-HIV therapeutics can be used in double or triple combinations such as AZT, DDI and nevirapine combinations.

In some embodiments, the agent encapsulated within the lipid particle may be, for example, the agent disclosed on pages 14-20 of US patent application Ser. No. 11 / 832,891, which application is incorporated herein by reference in its entirety. . The lipid particles can deliver an encapsulated agent inside the lumen of the ER upon fusion of the lipids of the ER membrane with the lipid particles.

Labeled lipids

In some embodiments, the lipid particle may comprise one or more labeled lipids labeled with one or more labels, such as a radiolabel, a fluorophore label, or a biotin label, thus allowing the particle itself to be labeled.

Labeled lipid particles can be used to specifically label the ER membrane of a cell, which can then be imaged. The type of cells that can be imaged by this technique is not specifically limited. Imaging can be performed, for example, by live or fixed imaging. Fixed imaging may refer to imaging of dead cells that may be immobilized with an immobilization medium, such as paraformaldehyde. The cells become permeable and can be probed with antibodies to detect specific proteins or labels and then mounted and imaged. For live cell microscopy, the cells may be still alive in the medium while imaging is performed.

Labeled particles can be used to label viruses. Examples of viruses that can be labeled using this approach include ER-budding viruses such as BVDV and HCV. If the label is a fluorophore label, the labeled lipid bilayer particles can be used for imaging of the labeled virus, which can be live and / or stationary imaging. If the label is a biotin label, labeled lipid particles can be used for purification of labeled viral particles. In some cases, such purification may be performed using streptavidin. Streptavidin can be linked to sepharose beads for batch purification of biotin-labeled material.

The invention is further illustrated by the following examples, but is not limited to the following examples in any way.

Example

1. Liposomal Formulations

Liposomes were freshly prepared for all assays described. A chloroform solution of lipids was placed in a glass tube and the solvent was evaporated under a stream of nitrogen gas. Unless stated otherwise, the lipid film was hydrated in 1 × PBS buffer to a final lipid concentration of 5 mM. The resulting multilayer plate vesicles were extruded 11 times through a polycarbonate filter of 100 nm pore diameter using a small extruder device. Liposomes were filter sterilized using a 0.22 μm filter unit. 1 (A) -1 (E) show the lipids used in these studies: A. DOPE; B. DOPC; C. CHEMS; D. PI; E. PS. The PEG-PE used in the experiment was PEG (MW-2000) -distearoylphosphatidylethanolamine. All lipids except cholesteryl hemisuccinate were purchased from Avanti Polar Lipids (USA) as all materials for the preparation of liposomes. Cholesteryl hemisuccinate was purchased from Sigma (UK).

2. Liposomes containing PI and / or PS are localized to ER.

The purpose of this experiment was to treat Huh7.5 cells (human cells) with liposomes containing PE and PC or CHEMS (with or without PI and / or PS lipids) to determine co-localization with the ER membrane. It was. Liposomes were labeled by incorporation of rhodamine-tagged PE (Rh-PE) into all liposomes. ER membranes of Huh7.5 cells were labeled using anti-EDEM antibodies. EDEM antibodies were purchased from Santa Cruz Biotechnology (USA). Co-localization was determined by confocal microscopy. Significant co-localization could serve as evidence that liposomes fuse with the ER membrane of Huh7.5 cells.

2.1 Specific Methods for Visualizing Liposome Co-localization with ER Membrane of Liposome-treated Huh7.5 Cells

Lipid composition PE: CH, PE: PC, PE: CH: PI, PE: PC: PI, PE: CH: PS, PE: PC: PS, PE: CH: PI: PS and PE: PC: PI: PS Liposomes having were prepared as described above and included 1% (total moles) of Rh-PE for visualization. Huh7.5 cells were allowed to adhere overnight on a number 1.5 glass cover slide, then the medium was exchanged and replaced with fresh medium containing liposomes added at a final lipid concentration of 50 μM. After 5 minutes incubation at 37 ° C./5% CO ₂ , remove the medium containing liposomes, wash the cells twice with 1 × PBS, incubate for another 30 minutes in fresh medium, then 1 × PBS / 0.1% Tween. Fix for 15 min in 4% paraformaldehyde diluted to -20 and wash twice in 1xPBS / 0.1% Tween-20. Cells are then incubated in 1 × PBS / 0.1% Tween-20 containing 4 μg / ml anti-EDEM antibody for 1 hour, washed twice in 1 × PBS / 0.1% Tween-20, 4 μg / ml FITC-labeled Incubate for 1 hour in 1 × PBS / 0.1% Tween-20 containing secondary antibody isolated and wash twice more. Cells were stained with DAPI and then mounted on microscope slides. Confocal images were taken using a Carl Zeiss LSM microscope and image analysis was performed using LSM software v5.10. 1F shows the structure of Rh-PE lipids used in these assays.

2.2 Co-localization of different liposomes with ER marker EDEM in Huh7.5 cells:

2 (A) -2 (F) show that liposomes containing lipid PI and / or PS co-localized with the ER-membrane protein EDEM. Liposomes were incubated with Huh7.5 cells for 5 minutes, then the medium was exchanged and the cells were incubated in liposome-free medium. After 30 min incubation the cells were fixed and probed with anti-EDEM antibody (green, top right image) and co-localization with Rh-PE lipids from liposomes (red, bottom left image) by confocal microscopy. Decided. DAPI (blue, top left image) was used as nuclear stain. Co-localization was measured by the presence of yellow color in the integrated image (bottom right). The experiment was repeated three times and representative images were shown. Figure 2A. PE: CH (molar ratio 3: 2) liposomes; Figure 2B. PE: PC (3: 2) liposomes; Figure 2C. PE: CH: PI (3: 1: 1) liposomes; 2D. PE: PC: PI (2: 2: 1) liposomes; Figure 2E. PE: CH: PS (3: 1: 1) liposomes; 2F. PE: PC: PS (2: 2: 1) liposomes; Figure 2G. PE: CH: PI: PS (3: 1: 0.5: 0.5) liposomes; 2H. PE: PC: PI: PS (1.5: 1.5: 1: 1) liposomes.

Images obtained as described above were used to quantify co-localization of ER membrane markers (EDEM) and liposomes.

2.3. Quantitative method of imaging co-localization:

The percentage of co-localization was measured using MetaMorph software (v.7, Molecular Devices, Downingtown, Pa.). Filter the images using a median filter set to 3 x 3 pixels, and average the thresholds used to determine the co-localization between the two images (rh-PE / red and EDEM / green). Intensity + 1 standard deviation (SD) was set. Reported values represent mean ± SD of 30 cells.

2.4. Results of co-localization percentage analysis of liposomes with ER markers (EDEM):

Quantitative results of image co-localization may suggest that incorporation of 20% PI or 20% PS into DOPE: CH or DOPE: DOPC liposomes significantly increases co-localization with ER membranes. Liposomes comprising DOPE: CH: PI and DOPE: CH: PS were 52% (SD = 8.0%) and 46% (SD = 8.1%) as compared to 13% (SD = 6.6%) for DOPE: CH alone ) Co-localization. Similarly, the compositions of DOPE: DOPC: PI and DOPE: DOPC: PS were 64% (SD = 8.1%) and 48% (SD ==) compared to 12% (SD = 4.7%) for DOPE: DOPC liposomes, respectively. 7.6%) co-localization. DOPE: CH: PI: PS liposomes showed 76% (SD = 8.7%) ER membrane co-localization and 88% (SD = 3.5%) co-localization was observed in DOPE: DOPC: PI: PS liposomes As such, the combination of 20% PI and 20% PS in DOPE: CH and DOPE: DOPC liposomes further increased co-localization to the ER membrane.

FIG. 3 shows the calculated co-localization of liposome-delivered rh-DOPE with EDEM antibodies determined by analyzing 30 individual cells per liposome preparation using Metamorph software, where the percentage of co-localization was determined. The threshold used was set to mean intensity + 1 SD for each image. Results shown represent mean co-localization and SD for 30 cells.

These results demonstrated that only liposomes containing lipid PI and / or PS in combination with PE showed increased co-localization with ER markers in Huh7.5 cells after 5-minute wave and 30-minute tracking with liposomes. . Since ER liposomes, ie liposomes containing PI and / or PS lipids, exhibit significant co-localization with ER markers, fluorescently-labeled ER liposomes have been used as a rapid and inexpensive technique for labeling ER membranes in eukaryotic cells. Can be used.

3. Lipids delivered via ER liposomes are incorporated into the envelope of a virus known to assemble and emerge from the ER membrane.

The purpose of the following experiments was to co-administer Madin-David Bovine Kidney (MDBK) cells and HCV cell culture (HCVcc) -infected Huh7.5 cells infected with bovine viral diarrhea virus (BVDV) with ER membranes by confocal microscopy. Treatment with liposomes shown to be localized and to find incorporation of tagged liposome lipids in secreted viral particles. BVDV and HCV are both viruses that assemble and emerge from the ER membrane; The incorporation of tagged lipids delivered via liposomes into secreted viral particles suggested fusion of liposomes with ER membranes of these cells. HIV-1-infected peripheral blood mononuclear cells (PBMCs) were used as controls to detect the incorporation of lipids into the budding virus from the plasma membrane.

3.1. Specific methods for monitoring the incorporation of liposome lipids into secreted viral particles using biotinylated PE lipids:

BVDV Cell Culture: Madin Darby Small Kidney Cells (MDBK) cells were seeded at 3 × 10 ⁵ cells per well of 6-well plate in complete DMEM / 10% FBS and ncp BVDV strain Pe515 (NaI) with an infection multiplicity (MOI) of 0.1 Animal Disease Laboratory, UK) and passed through 2 ml of fresh RPMI 1640 medium containing 10% (vol / vol) fetal bovine serum at a 1: 8 dilution every 3 days. Liposomal treatment was initiated after achieving stable infection (determined by RT-RCR) to quantify secreted BVDV particles. Quantitative PCR was performed on 500 μl of supernatant using the QIAamp virus RNA purification kit (QIAGEN) according to the manufacturer's protocol. Real-time PCR was performed using SyBr Green Mix (Qiagen) and primers oriented against ncp BVDV RNA (Forward: TAG GGC AAA CCA TCT GGA AG, Reverse Primer: ACT TGG AGC TAC AGG CCT CA).

JC-1 HCV Cell Culture (HCVcc): Huh7.5 cells (Apass, LLC, St. Louis, USA) were loaded with complete DMEM (100 U / ml penicillin, 10% fetal bovine serum (FBS) 100 μg / ml streptomycin, 2 mM L-glutamine and 1 × MEM). All incubations were performed at 37 ° C./5% CO ₂ . Cells were infected for 1 hour at MOI = 0.5 and liposome treatment was initiated if more than 50% of the cells were tested positive for HCVcc infection (determined by HCV core protein immunofluorescence). Quantification of viral RNA from the supernatant, as well as infectivity of secreted particles, was determined using quantitative PCR and core protein immunofluorescence, respectively.

HIV cell culture: Peripheral blood mononuclear cells (PBMCs) from four uninfected donors using Histopaque density gradient centrifugation (Sigma-Aldrich, Gillingham, UK) Isolation, pooling, phytohemagglutinin (PHA, 5 μg / ml) in complete RPMI (RPMI + 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin and 2 mM L-glutamine) before starting the experiment ) And then for 72 hours with interleukin-2 (IL2, 40 U / ml). All incubations were performed at 37 ° C./5% CO ₂ unless otherwise stated here. To infect the cells, 100 TCID ₅₀ (tissue culture infection 50%) of ⁴ × 10 ⁶ PHA-activated PBMCs and primary isolate stocks were incubated together in 2 ml complete RPMI / 10% FBS per well in 6-well plates. . Cells were infected for 16 hours and washed three times with complete RPMI medium before incubation with liposomes.

Purification of Secreted Biotin-labeled Particles: Virus-infected cells are grown in 75 cm 2 flasks, and then the medium is replaced with medium containing 50 μM b-PE-labeled 22: 6 ER liposomes and incubated for 48 hours. It was. Cells were then washed twice in PBS and incubated in fresh medium containing no liposomes for an additional 24 hours. Supernatants containing secreted particles were harvested, cells were counted using trypan blue staining, and the supernatants were normalized to sample cell numbers using PBS. Secreted HCVcc and BVDV were titrated by quantitative PCR and the infectivity of secreted virions was determined. HIV-1 was quantified by p24 capture ELISA. High performance streptavidin sepharose (GE Healthcare) was used to capture biotinylated particles. Sepharose beads were washed twice by diluting 1:50 (vol: vol) in PBS and gently mixing for 5 minutes at room temperature and pelleted by centrifugation at 1500 rpm for 3 minutes. Sepharose is resuspended to form a 50% slurry in PBS and added to the culture supernatant (200 μl 50% slurry per 10 ml of culture supernatant). After incubating the Sepharose and the supernatant at room temperature for 1 hour with gentle shaking, the Sepharose beads were washed five times in PBS as described above. To quantify the amount of b-PE-labeled virion, take 1 ml of culture supernatant, 500 μl of which is used for total virus quantification, 500 μl captured on streptavidin sepharose, and in PBS Washed five times and used directly for RNA quantification by incubating the beads with viral RNA lysis buffer (Qiagen) for HCVcc and BVDV RT-PCR analysis, or by incubating in 1% ampogen for the p24 HIV ELISA assay. .

3.2. Incorporation of b-PE lipids into secreted viral particles:

1G shows the structure of the biotinylated PE lipids (b-PE) used.

ER liposomes, ie liposomes containing PI and / or PS lipids, incorporate biotin-labeled PE (b-PE) at 0.1, 0.5, 1, 5 or 10 mol% and secreted HCV and BVDV (two types The optimal concentration for tagging of ER-emergence virus was determined to be 1%, which captured 90% (SD = 3.6%) and 91% (SD = 1.5%) of the total number of secreted virions, respectively ( 4). PBMCs infected with primary isolates of HIV-1 (LAI) were also treated with b-ER liposomes and the secreted HIV-1 particles did not contain detectable amounts of tagged lipids (FIG. 4). These results can underscore the specificity of this system for delivering lipids to ER and ER-associated membranes because reproductive HIV-1 assembly occurs in the plasma membrane.

4 shows ER liposomes containing b-PE lipids incubated for 48 hours with JC-1-infected Huh7.5 cells, BVDV-infected MDBK cells or HIV-1-infected PBMCs (final lipid concentration of 50 μM). The experimental results are shown. Infected cells were washed and b-PE-labeled virus particles secreted during the subsequent 24-hour incubation period in the absence of liposomes were captured using streptavidin-sepharose resin. The results are expressed as the percentage of tagged viral particles captured by streptavidin relative to the total amount of virion secreted (100%) in the same sample.

The results in FIG. 4 show that lipids delivered to BVDV-infected MDBK cells and HCVcc-infected Huh7.5 cells via ER-localized liposomes (liposomes comprising PE in combination with PI and / or PS) are mostly BVDV and HCVcc. It can be demonstrated that it exists in the viral envelope but not in the HIV envelope secreted during liposome treatment. BVDV and HCV are known to assemble and sprout from the ER membrane, while HIV assembles and emerges from the plasma membrane, which means that liposomes containing PI and / or PS lipids can fuse with the ER membrane of the cell. It is a further increase.

The incorporation of tagged lipids into ER-germinated virus after treatment with ER liposomes cannot be limited to biotinylated lipids, but fluorescence to generate virions containing fluorescent lipids for visualization by fluorescence microscopy. Lipids may also be used.

3.3. Imaging method of rh-PE-tagged HCVcc after rh-PE liposome treatment using confocal microscopy:

After Huh7.5 cells were grown to full confluence in 75 cm 2 flasks, the medium was replaced with medium containing 50 μM rh-PE-labeled 22: 6 ER liposomes. Cells were incubated for 48 hours, washed twice in PBS, and then incubated for 24 hours in fresh medium without liposomes. Supernatants containing secreted particles were harvested. Secreted HCVcc was titrated by quantitative PCR and the infectivity of secreted virions was determined as described above. For visualization of rh-HCVcc by confocal microscopy, uninfected Huh7.5 cells were allowed to adhere overnight on a number 1.5 glass cover slide in complete DMEM / 10% FCS, and then the medium was replaced with rh-HCVcc virus stock. And incubated for 1 hour. After infection, cells are washed twice with PBS, fresh medium is replaced for various incubation times, washed twice with 1 × PBS, fixed in methanol: acetone (1: 1, vol: vol) for 10 minutes, and finally Wash twice in 1 × PBS / 0.1% Tween-20. Cells are then incubated for 1 hour in 1 × PBS / 0.1% Tween-20 containing primary antibody, washed four times in 1 × PBS / 0.1% Tween-20, and 1 × PBS / 0.1% containing fluorescently labeled secondary antibody. Incubate for 1 hour in Tween-20 and wash 4 more times. Cells were stained with DAPI and then mounted on microscope slides. Confocal images were taken using a Carl Zeiss LSM microscope and image analysis was performed using LSM software v5.10.

3.4. Visualization of rh-PE-tagged HCVcc by confocal microscopy:

HCVcc infection was visualized in Huh7.5 cells using 1% rh-ER liposomes and fixed cell confocal microscopy (FIG. 5). Rh-tagged HCVcc was collected for 24 hours after 48 hours incubation in the presence of 1% rh-ER liposomes and used to infect uninfected cells at MOI = 0.1 for 1 hour. Fixed confocal images were taken immediately after 1 hour infection as well as after 6 and 24 hours of infection, and the cells permeable to probe positive HCVcc particles were probed with anti-HCV core antibody. In these images, the core-positive particles appeared as a single cluster of approximately 1 μm in diameter on the cell surface until after 1 hour of infection, at which point the cluster appeared to be endocytosis and diffused into clusters of approximately 5 μm. This large cluster migrated toward the nucleus of the cell, forming a characteristic depression of the nucleus of the infected cell (FIG. 5), at which point the cluster dispersed and rh-tagged lipids began to separate from the HCV core protein. . Increased levels of core protein were observed in cells after approximately 24 hours of infection and could indicate established infection and de novo core protein synthesis.

FIG. 5 shows Rh-PE-tagged JC- incubated with uninfected Huh7.5 cells for 1 hour (MOI = 0.1), followed by washing the cells and incubating for additional 0, 6 or 24 hours in fresh medium. Experimental results for 1 HCVcc (red, bottom-left panel) are shown. After each incubation time, cells were fixed, stained with anti-HCV core antibody (green, top-right panel) and DAPI (blue, top-left panel), mounted on microscope slides and confocal microscopy images. The integrated image is shown in the bottom-right panel. Representative images from each incubation period are shown. Although fixed cell confocal microscopy was used in these assays, this technique provides a method for labeling virions with a wide selection of lipid-fluorophore conjugates for tracking by live cell microscopy. This type of incorporation technique is not limited to biotin or fluorescently tagged lipids, as other lipid conjugates or transmembrane proteins can also be incorporated into ER liposomes for specific delivery of cells to the ER membrane.

4. Lipids delivered through ER liposomes have a longer lifespan in cells compared to pH-sensitive liposomes.

The purpose of this experiment was to treat MDBK cells with fluorescently-labeled liposomes to monitor influx and incorporation into the cell membrane over time. pH-sensitive liposomes, ie DOPE-CHEMS or DOPE-CHEMS-PEG-PE liposomes that do not contain PI and PS lipids, can be considered to enter the cell, and after disruption of the liposome membrane in the endosome, the lipid continues to lysosomes. It was believed to follow the endosomal route of. If liposomes containing PI and / or PS lipids can be fused with other membranes in the cell, it is certain that they have a longer lifespan compared to pH-sensitive liposomes. Rho-PE lipids delivered to cells via liposomes were visualized by fluorescence microscopy over a period of 48 hours after 5 minutes treatment with MDBK cells.

4.1. Specific Methods for Monitoring Liposomal Incorporation into Cell Membranes

PE: CH, PE: CH: PI and PE: CH: PS liposomes were prepared as described above and included 1% (total moles) of Rh-PE for visualization. MDBK cells were seeded at 50% confluence on 6 well plates and attached overnight. Cells were washed twice in 1 × PBS and then treated at 37 ° C., 5% CO ₂ for 5 minutes with Rh-labeled liposomes added to 2 ml of complete RPMI at a final lipid concentration of 50 μM. After 5 minutes incubation, cells were washed twice in 1 × PBS, 2 ml of fresh complete RPMI medium was added to each well and plates were incubated for 1, 10, 24 and 48 hours. At the end of each incubation time, cells were washed twice and then fixed in 4% paraformaldehyde diluted in 1 × PBS / 0.1% Tween-20 for 15 minutes and washed twice in 1 × PBS / 0.1% Tween-20. Cells were imaged after staining with DAPI. Fluorescence images were taken using a Nikon Eclipse TE2000-U microscope and image analysis was performed using Nikon ACT-1 software v2.70.

4.2. Incorporation of liposomes into cell membranes:

6 (A) -6 (C) show that fluorescence microscopy images of liposomes comprising lipid PE in combination with PI or PS showed increased incorporation into cell membranes as compared to pH-sensitive liposomes. MDBK cells were treated with Rh-PE labeled liposomes for 5 minutes, then the cells were washed and incubated in medium for only 1, 10, 24 and 48 hours. After each incubation time, cells were fixed and Rh-PE lipids (red) were visualized under fluorescence microscopy. DAPI (blue) was used as nuclear stain. The experiment was repeated twice and representative images from one experiment were shown. 6A. PE: CH (molar ratio 3: 2) liposomes. 6B. PE: CH: PI (molar ratio 3: 1: 1) liposomes. Figure 6C. PE: CH: PS (molar ratio 3: 1: 1) liposomes.

The results in FIG. 6 show that liposomes comprising PE in combination with PI or PS can be incorporated into the membrane of MDBK cells. Rh-PE lipids delivered to cells via PE: CH lipids disappeared almost 24 hours after removal of liposomes from the cell medium, while lipids delivered via PE: CH: PI and PE: CH: PC exceeded 48 hours. It was still present in the cells, suggesting more incorporation into the membrane.

4.3. Quantification of Liposomal Influx and Lipid Retention in Treated Cells:

To monitor the liposome influx rate in Huh7.5 cells over a 4-day incubation period, cells were treated with DOPE: CH and DOPE: DOPC: PI: PS containing 1% rh-PE with a final lipid concentration of 50 μM in medium. Incubate with liposomes. Cells were seeded at low density and liposome uptake was measured for cell growth. After 4 days incubation, the treated Huh7.5 cells were washed and returned to medium without any liposomes, resulting in half-life of rh-DOPE lipids delivered via DOPE: CH and DOPE: DOPC: PI: PS liposomes. Monitoring.

4.4. Methods of Quantifying Liposomal Influx and Lipid Retention in Treated Cells:

Liposomes were prepared as described above and included 1% (total moles) of rh-PE to monitor influx in cells. For long-term (4 days) liposome uptake assays, Huh7.5 cells were seeded on 6 well plates at 10 ⁵ cells / well in 2 ml of complete DMEM medium / 10% FBS. Rh-PE-labeled liposomes were added to the cells at a final phospholipid concentration of 50 μM and incubated at 37 ° C./5% CO ₂ for 2, 24, 48, 72 and 96 hours. After incubation time, cells were harvested and analyzed. For analysis, cells were washed twice in 1 × PBS, counted, resuspended in 200 μl 1 × PBS / 0.5% Triton X-100, transferred to 96 well plates, and spectrofluorometer at λex = 550 nm and λem = 590 nm. Read on To measure the retention of rh-PE lipids inside Huh7.5 cells after the 96 hour incubation described above, the cells were washed three times in 1 × PBS, the medium was replaced with fresh DMEM / 10% FBS, and the cells were added 8, 24 Incubated for 30 and 48 hours. After incubation time, cells were harvested and analyzed as described above.

4.5. Results of Liposomal Influx and Lipid Retention Assay in Huh7.5 Cells:

As shown in FIG. 7, actively dividing Huh7.5 cells showed continuous influx of DOPE: DOPC: PI: PS liposomes over a 4 day incubation period. On day 4, DOPE: DOPC: PI: PS-treated cells showed fluorescence of 1.5 × 10 ⁻³ AU / cells (SD = 3.4 × 10 ⁻⁴ AU / cells), which was observed in DOPE: CH liposome treatment. 6 times greater than (2.5 × 10 ⁻⁴ AU / cell (SD = 5.5 × 10 ⁻⁵ AU / cell)). In fact, the maximum fluorescence observed in DOPE: CH-treated cells was reached only after a 24 hour treatment period (5.0 × 10 ⁻⁴ AU / cell (SD = 1.1 × 10 ⁻⁴ )), after which the cell-associated fluorescence It decreased slowly, suggesting a reduced liposome influx or increased outflow of rh-PE lipids or both.

Based on these experiments, rh-DOPE lipids from DOPE: CH liposomes showed a half-life in cells approximately 7 hours after removal of liposomes from the medium. For cells treated with DOPE: DOPC: PI: PS liposomes, the rh-DOPE half-life was extended to approximately 29 hours, suggesting greater incorporation of these liposomes into the membranes of treated cells.

7 shows experimental results for ER-targeted liposomes showing increased intracellular inflow and lipid retention inside Huh7.5 cells. Rh-labeled liposomes (50 μM final lipid concentration) were incubated with Huh7.5 cells for 4 days (96 hours). Liposomal influx into cells was monitored over the incubation period and DOPE: CH (red, solid line) and DOPE: for maximum value (1.5 × 10 ⁻³ AU / cell, DOPE: DOPC: PI: PS liposome, 96 hours read): Shown as fluorescence observed per cell for both DOPC: PI: PS liposomes (black, solid line). Fluorescence was measured at λex = 550 nm and λem = 590 nm. After 96 hours of incubation, cells were washed and placed in fresh medium (without liposomes) to monitor the retention of rh-DOPE lipids in the cells over an additional 48 hours. DOPE: CH (red, dotted line) and DOPE: DOPC: PI: PS liposomes for maximum (2.4 × 10 ⁶ cells / ml, DOPE: DOPC: PI: PS liposomes, 72 hours readout) and DOPE: DOPC: PI: PS liposomes (black) during the incubation period. , Dotted lines). The data represent the mean and SD of three samples from three independent experiments.

5. ER liposomes show increased stability and intracellular influx in the presence of serum:

The general use of liposomes as drug delivery systems has been hampered by several problems. Among others, leakage of liposome contents mediated by serum proteins. Calcein-encapsulated liposomes were used to monitor the stability of liposomes in cell-free medium containing 10% FBS over a 4 day period. Calcein is a water soluble self-quenching fluorophore that remains quenched when encapsulated within liposomes; Liposomal destabilization will lead to leakage of fluorescence and subsequent dequenching.

5.1. Methods for quantifying liposome stability and intracellular influx in FBS:

In order to monitor the stability of liposomes in the presence of 10% FBS, calcein-loaded liposomes are prepared, separated from unencapsulated calcein by size-exclusion chromatography, and complete DMEM / 10% FBS in the absence of cells. To a final phospholipid concentration of 50 μM. Liposomes are incubated for 4 days and samples of liposome-containing medium are taken every 24 hours to monitor calcein dequenching at λex = 490 nm and λem = 520 nm as a result of liposome destabilization and leakage of calcein into the surrounding medium. It was. Addition of Triton X-100 at a final concentration of 1% after 4 days incubation achieved 100% calcein dequenching to disrupt the liposome membrane to correct the fluorescence scale:% leak = ((I _n -I ₀ ) / ( I ₁₀₀ −I ₀ )) × ₁₀₀ where I ₀ is fluorescence at 0 h, I _n is fluorescence at n and I ₁₀₀ is total dequenched calcein fluorescence after addition of Triton.

For intracellular inflow assays, liposomes were prepared as described above and included 1% (total moles) of rh-PE to monitor inflow into cells. For short-term liposome inflow assays with or without serum, in 6-well plates at a final phospholipid concentration of 50 μM in serum-free complete DMEM, or complete DMEM supplemented with 10% FBS or 10% human serum (Sigma). Rh-PE-labeled liposomes were added to Huh7.5 cells grown until full growth and incubated for 24 hours. After incubation, cells were washed twice in 1 × PBS, counted, resuspended in 200 μl 1 × PBS / 0.5% Triton X-100, transferred to a 96 well plate and placed on a spectrophotometer at λex = 550 nm and λem = 590 nm. Read.

5.2. Results of quantitative assay of liposome stability and intracellular influx in the presence of 10% serum:

8A shows calcein release rate from inside pH-sensitive DOPE: CH and ER-targeted DOPE: DOPC: PI: PS liposomes. After 4 days incubation, 58% of calcein (SD = 12.6%) was released from DOPE: PE liposomes, whereas only 32% of calcein (SD = 9.2%) leaked from DOPE: DOPC: PI: PS liposomes. This suggests greater stability in the presence of serum.

To monitor the effect of FBS and human serum on the influx of liposomes into Huh7.5 cells, DOPE: CH and DOPE: DOPC: PI: PS liposomes containing 1% rh-PE in the membrane were prepared and serum- Incubate with Huh7.5 cells (final liposome concentration of 50 μM) for 24 hours in the absence of medium and medium containing 10% FCS or 10% human serum. Liposomal influx in cells was expressed as the amount of fluorescence per cell (arbitrary unit, AU) after the 24-hour incubation period. A significant decrease in DOPE: CH liposome influx was observed in the presence of FBS compared to serum free medium (5.0 × 10 ⁻⁴ AU / cell (SD = 7.0 × 10 ⁻⁵ AU / cell, respectively) vs. 8.2 × 10 ⁻⁴ AU / Cells (SD = 2.1 × 10 ⁻⁴ AU / cell), P = 0.02, FIG. 8B). There was no significant difference in the presence of human serum. In contrast, DOPE: DOPC: PI: PS liposomes serum showed a significant increase of the influx in the presence of FBS-free medium as compared to the (each 6.6x10 ^-4 AU / cell (SD = 8.4x10 ^-5 AU / cells) versus 3.1x10 ^-4 AU / cell (SD = 1.2x10 ^-4 AU / cell), P = 0.003, Fig. 8b). In addition, the presence of human serum significantly increased the efficiency of DOPE: DOPC: PI: PS liposome influx in Huh7.5 cells compared to FBS (1.4 × 10 ⁻³ AU / cell (SD = 1.8 × 10 ⁻⁴ AU / cell). ), P = 0.001, FIG. 8B).

10A-10B show experimental results demonstrating that ER-targeted liposomes have increased stability and intracellular influx in the presence of serum. (A) Self-quenching calcein-loaded liposomes (final lipid concentration of 50 μM) were incubated in complete DMEM + 10% FBS and incubated at 37 ° C. for 4 days. Every 24 hours, a sample of the culture was used to measure calcein dequenching at λex = 485 nm and λem = 520 nm. The results are expressed as the percentage of calcein released from liposomes compared to the maximum fluorescence determined by the addition of Triton X-100 to disrupt the liposome membrane at the end of the incubation period. (B) Rh-labeled liposomes (50 μM lipid concentration) were incubated with Huh7.5 cells for 24 hours in the presence of 10% bovine serum (FBS), 10% human serum or in serum-free medium. After incubation time, cells were harvested, counted and fluorescence was measured at λex = 550 nm and λem = 590 nm. The results are shown as the average fluorescence measured per cell for each sample. All data represent mean and SD of three samples from three independent experiments.

These studies using DOPE: DOPC: PI: PS liposomes may suggest that such phospholipid combinations may exhibit more desirable interactions with both cells and serum compared to DOPE: CH liposomes. In the presence of 10% FBS, DOPE: DOPC: PI: PS liposomes showed 45% less leakage of encapsulated content after 4 days incubation compared to DOPE: CH liposomes. DOPE: DOPC: PI: PS liposomes also showed increased influx into Huh7.5 cells in the presence of FBS, which was further increased in the presence of human serum. In contrast, DOPE: CH liposome influx appeared to be inhibited in the presence of FBS as compared to serum-free medium. The present invention is not limited by its theory of operation, but these results indicate that liposomes targeting ER, ie, liposomes containing PI and / or PS lipids, are endo lysed by different cellular receptors than the receptors used by DOPE: CH liposomes. It may be suggested that cytosine and endocytosis through this mechanism can be enhanced by the presence of serum.

6. Cytotoxicity of ER Liposomes in Huh7.5 Cells and PBMCs

The purpose of these experiments was to determine the effect of liposomes on cell viability over one circulating treatment (5 days) by both Huh7.5 cells and PBMC.

6.1. Specific methods for determining cytotoxicity in Huh7.5 cells and PBMCs:

Lipid composition PE: CH (3: 2), PE: PC (3: 2), PE: PI (3: 2), PE: CH: PI (3: 1: 1), PE: PC: PI (1.5: 1.5: 2), PE: PS (3: 2), PE: CH: PS (3: 1: 1), PE: PC: PS (1.5: 1.5: 2), PE: PI: PS (3: 1: 1), liposomes with PE: CH: PI: PS (3: 1: 0.5: 0.5) and PE: PC: PI: PS (1.5: 1.5: 1: 1) were prepared as described above. Huh7.5 cells and PBMC were seeded in 96 well plates, respectively, in 200 μl of complete DMEM and RPMI + IL2 medium at a concentration of 5 × 10 ⁴ cells / well, and of liposomes encapsulating 1 × PBS with final lipid concentrations ranging from 0 to 500 μM. Incubate in the presence. After 5 days incubation, cell viability was determined according to the manufacturer's protocol by MTS-based cell proliferation assay (CellTiter 96®, Promega, San Luis Obispo, USA).

6.2. Cytotoxicity in Huh7.5 cells and PBMC when treated with PBS liposomes for 5 days:

9 shows viability of Huh7.5 cells after 5 days incubation with different liposome formulations encapsulating 1 × PBS. Final lipid concentrations in the medium ranged from 0 to 500 μM. The results represent the mean value of three samples from three independent experiments.

10 shows the viability of PBMCs after 5 days incubation with different liposome formulations encapsulating 1 × PBS. Final lipid concentrations in the medium ranged from 0 to 500 μM. The results represent the mean value of three samples from three independent experiments.

The results in FIGS. 9 and 10 may indicate that only liposomes containing lipid CHEMS are cytotoxic in Huh7.5 cells and PBMC when added to cells at concentrations greater than 60 μM. ER liposomes that do not contain this lipid show little cytotoxicity, if any, compared to pH-sensitive liposomes (PE: CH) and are therefore preferred for in vivo use.

7. Secretion of HIV-1 from Infected PBMCs Treated with ER-Liposomes

The purpose of these experiments was to monitor changes in levels of HIV-1 secretion from HIV-1-infected PBMCs treated with different liposome compositions.

7.1. Specific methods for single-circulating HIV secretion assays:

Lipid Composition PE: CH (3: 2), PE: PC (3: 2), PE: PI (3: 2), PE: CH: PI (3: 1: 1), PE: PS (3: 2) Liposomes with PE: CH: PS (3: 1: 1), PE: CH: PI: PS (3: 1: 0.5: 0.5) and PE: PC: PI: PS (1.5: 1.5: 1: 1) Was prepared as described above. Determination of PBMCs stimulated as indicator cells and p24 antigen production as endpoints was used to assess changes in the secretion of HIV as a result of infection with virions secreted from drug-treated cells. PBMCs from four normal (uninfected) donors were isolated, pooled, and fully RPMI (RPMI + 10% heat—using histopark density gradient centrifugation (Sigma-Aldrich, Gillingham, UK). Inactivated FBS, 100 U / ml penicillin, 100 μg / ml streptomycin and 2 mM L-glutamine) with phytohemagglutinin (PHA, 5 μg / ml) for 48 hours, followed by interleukin-2 (IL2, 40 U / ml) for 72 hours. Unless stated otherwise, all experiments were performed in 96-well microtiter plates and all incubations were performed at 37 ° C./5% CO ₂ . To infect the cells, 100 TCID ₅₀ (tissue culture infection 50%) of ⁴ × 10 ⁵ PHA-activated PBMCs and primary isolate stocks were added to each well. After 16 hours overnight incubation, cells were washed three times with complete RPMI medium and resuspended in complete RPMI / IL2 containing appropriate free drug or liposome treatment (final lipid concentration of 50 μM). On day 5, supernatants containing HIV virions secreted from drug-treated cells were collected and p24 concentrations were quantified for each by p24 capture ELISA.

7.2. Results from single-circulating HIV secretion assays:

11 shows secretion of HIV from infected PBMCs during treatment with liposomes for 5 days. All liposomes encapsulated 1 × PBS solution, which was added to the cell culture medium at a final lipid concentration of 50 μM. Virus secretion was calculated according to the quantification of HIV core protein, p24, in supernatants of treated and untreated PBMCs by capture ELISA. Results are expressed as percentage of HIV secretion compared to the untreated control and represent the mean of three samples from two independent experiments. Assays were performed on three genetically diverse isolates of HIV-1 including LAI (clade B), 93UG067 (clade D) and 93RW024 (clade A).

The results in FIG. 11 can demonstrate that ER liposomes containing lipid PI can reduce HIV secretion from PBMCs by approximately 20% compared to untreated controls. Non-ER targeting liposomes (PE: CH and PE: PC) and ER liposomes that do not contain PI lipids had no effect on HIV secretion.

8. Infectivity of HIV-1 Secreted from Infected PBMCs Treated with ER Liposomes

The purpose of these experiments was to monitor the infectivity of HIV-1 virions secreted from HIV-1-infected PBMCs treated with different liposome compositions.

8.1. Specific methods for single-circulating HIV infectivity assays:

Supernatants containing HIV virions secreted from liposome-treated cells as described in the section above were used to determine the infectivity of HIV virions secreted from PBMCs treated with liposomes. All supernatants were diluted to a final p24 concentration of 10 ng / ml in complete RPMI / IL2, 100 μl was also added to ⁴ × 10 ⁵ PHA-activated PBMC for a final p24 concentration of 5 ng / ml at 100 μl of medium and overnight Incubated. The next day, cells were washed as described, resuspended in 200 μl of fresh RPMI / IL2, incubated for 4 days, and then the supernatant was collected and assayed by capture ELISA for p24 content.

8.2. Results from single-circulating HIV infectivity assays:

12 shows the infectivity of HIV virions secreted from liposome-treated HIV-infected PBMCs. Secreted virus particles were used to infect uninfected PBMCs, determine the ability to infect cells by removing the supernatant and leaving the cells untreated for 5 days before measuring viral secretion. The results are expressed as a percentage of HIV infectivity compared to the untreated control and represent the average of three samples from two independent experiments. Assays were performed on three genetically diverse isolates of HIV-1 including LAI (clade B), 93UG067 (clade D) and 93RW024 (clade A).

The results in FIG. 12 may demonstrate that certain ER liposomes can reduce the infectivity of virus particles secreted from treated PBMCs. The greatest antiviral activity was observed in ER liposomes comprising lipid CHEMS in combination with PI and / or PS, wherein the infectivity of viral particles was less than 20% of untreated virions. Non-ER liposomes (PE: CH and PE: PC) as well as ER liposome PE: PS had no effect on viral infectivity.

9. ER liposomes show more efficient intracellular content release compared to pH-sensitive liposomes.

In these experiments, rhodamine-labeled liposomes encapsulating the self-quenching concentration of the fluorescent molecule calcein were prepared and incubated in the presence of Huh7.5 cells. Since calcein is released into the intracellular space and dequenched, delivery of the encapsulated content in the cell was monitored by increasing fluorescence.

9.1 Specific Methods for Measuring Intracellular Delivery of Liposomes in Huh7.5 Cells:

For fluorescence assays, 5 × 10 ⁶ Huh7.5 cells were seeded in 25 cm 2 flasks overnight in complete DMEM / 10% FBS. The next day, calcein-loaded liposomes containing 1% rh-PE were added to the medium (final phospholipid concentration of 50 μM) and incubated at 37 ° C. or 4 ° C. for 30 minutes. After incubation, cells were washed twice in 1 × PBS, detached with trypsin / EDTA (Invitrogen), washed twice more and resuspended in 600 μl PBS. Fluorescence measurements were measured and averaged using three 200 μl aliquots. Calcein dequenching was measured at λex = 485 nm and λem = 520 nm and rhodamine fluorescence was measured at λex = 550 nm and λem = 590 nm. Incubation of the liposomes with the cells at 4 ° C. yielded an initial calcein to rhodamine fluorescence ratio of liposomes bound to the cells in the absence of endocytosis and was used to adjust the values at 37 ° C.

9.2. Intracellular Release of Encapsulated Calcein from Liposomes in Huh7.5 Cells:

Mean rh-DOPE fluorescence in Huh7.5 cells after 45 min incubation with liposomes reflects the influx of liposomes and mean calcein fluorescence indicates intracellular dequenching, and therefore the emission of fluorescent dyes. The calculated ratio of calcein to rhodamine fluorescence was taken as a measure of the amount of aqueous marker released in cells per cell-associated liposome. The calcein / rhodamine ratio for DOPE: CH liposomes was calculated to be 10.3 (SD = 2.6), while the ratio for DOPE: DOPC: PI: PS liposomes was 15.7 (SD = 2.4), ie an increase of 152% ( P = 0.02, FIG. 13).

FIG. 13 shows experimental results for self-quenching calcein-loaded rh-PE-labeled liposomes (final lipid concentration of 50 μM) incubated with Huh7.5 cells in full DMEM / 10% FBS for 45 minutes. . Intracellular dequenching of calcein from liposomes after incubation was measured at λex = 490 nm, λem = 520 nm, and total liposome influx was determined by fluorescence measurements at λex = 550 nm, λem = 590 nm during the same incubation period. . Assays were performed at 37 ° C. and 4 ° C. and all 4 ° C. values were subtracted from the 37 ° C. values to correct for liposome binding without endocytosis. The ability of liposomes to deliver encapsulated calcein into Huh7.5 cells was calculated by calculating the ratio of calcein dequenching and rh-PE fluorescence in treated cells after incubation. The data represent the mean and SD of three samples from three independent experiments.

The results in FIG. 13 show intracellular calcein release per liposome compared to PE: CH liposomes, a liposome composition specifically designed for efficient intracellular delivery of a compound in which a liposome comprising PE in combination with PI and / or PS is encapsulated It can be proposed to have an increased level of. In these assays, PE: PC: PI: PS liposomes showed 1.5-fold greater calcein release compared to PE: CH liposomes.

10. Secretion of HIV-1 from PBMCs Treated with Liposomes Encapsulating 1 mM NB-DNJ

The purpose of these experiments was to determine the ability of liposomes to deliver encapsulated iminosugars (ie, NB-DNJ) to HIV-infected PBMCs. Liposomes containing lipids PI and PS were compared to pH-sensitive liposomes (PE: CH) and pH-sensitive liposomes (PE: PC).

10.1. Specific methods for single-circulating HIV secretion assays:

Lipid Composition PE: CH (3: 2), PE: PC (3: 2), PE: PI (3: 2), PE: CH: PI (3: 1: 1), PE: PS (3: 2) Liposomes with PE: CH: PS (3: 1: 1), PE: CH: PI: PS (3: 1: 0.5: 0.5) and PE: PC: PI: PS (1.5: 1.5: 1: 1) Was prepared as described above except that all liposomes encapsulated 1 mM NB-DNJ in 1 × PBS. HIV secretion assay was performed as described above. Liposomes were purified by size-exclusion chromatography from unencapsulated NB-DNJ. The results in liposomes were compared with the results in NB-DNJ added at a final concentration of 1 mM in cell culture medium.

10.2 Results from Single-Circulating HIV Secretion Assay:

FIG. 14 shows secretion of HIV from free PBMC: free versus liposome-mediated delivery during 5 days treatment with 1 mM NB-DNJ. Liposomes encapsulated 1 mM NB-DNJ, which was added to the cell culture medium at a final lipid concentration of 50 μM. Virus secretion was calculated as described above. Results are expressed as percentage of HIV secretion compared to the untreated control and represent the mean of three samples from two independent experiments. Assays were performed on three genetically diverse isolates of HIV-1 including LAI (clade B), 93UG067 (clade D) and 93RW024 (clade A).

The results in FIG. 14 show that liposomes containing lipid PI or PS deliver antiviral NB-DNJ to HIV-infected PBMCs, resulting in similar (if not better) antiviral activity (reduction of HIV secretion compared to PE: CH liposomes). To be determined by the

11. Infectivity of HIV-1 Secreted from Infected PBMCs Treated with Liposomes Encapsulating 1 mM NB-DNJ

The purpose of these experiments was to determine the ability of liposomes to deliver encapsulated iminosugars (ie, NB-DNJ) to HIV-infected PBMCs. Liposomes containing lipids PI and PS were compared to pH-sensitive liposomes (PE: CH) and pH-sensitive liposomes (PE: PC).

11.1. Specific methods for single-circulating HIV infectivity assays:

The infectivity of secreted HIV virions from PBMCs treated with liposomes as described above was determined except that all liposomes encapsulated 1 mM NB-DNJ in 1 × PBS. Results in virions secreted from liposome-treated cells were compared with results from free NB-DNJ-treated and untreated cells.

11.2. Results from single-circulating HIV infectivity assays:

15 shows the infectivity of HIV virions secreted from NB-DNJ-liposomes or free NB-DNJ-treated HIV-infected PBMCs. The secreted virus particles were used to infect uninfected PBMCs and the ability to infect cells was determined as described above. The results are expressed as a percentage of HIV infectivity compared to the untreated control and represent the average of three samples from two independent experiments. Assays were performed on three genetically diverse isolates of HIV-1 including LAI (clade B), 93UG067 (clade D) and 93RW024 (clade A).

The results in FIG. 15 may demonstrate that treatment of HIV-infected PBMCs with ER liposomes encapsulating 1 mM NB-DNJ reduces secretion and infectivity of HIV compared to untreated controls. Comparison of the results between the pH-sensitive liposomes, which are liposomes that do not contain PI and PS lipids, and the liposomes that contain lipids PI and PS, showed no significant difference in antiviral activity when encapsulating 1 mM NB-DNJ.

Antiviral activity can be further enhanced by chemically linking soluble forms of gp120 / gp41 targeting molecules such as CD4 to the outer surface of the drug-encapsulated liposomes. The targeting molecule should result in increased influx of drug-loaded liposomes into HIV-infected cells through receptor-mediated endocytosis in addition to neutralization of free virus particles to prevent infection.

12. Cytotoxicity of ER-Liposomes Encapsulating 1 mM NB-DNJ in PBMC:

The purpose of these experiments was to determine the effect of liposomes encapsulating 1 mM NB-DNJ on cell viability over one cycle of treatment with PBMC (5 days).

12.1. Specific methods for the determination of cytotoxicity in PBMC:

Lipid Composition PE: CH (3: 2), PE: PC (3: 2), PE: PI (3: 2), PE: CH: PI (3: 1: 1), PE: PS (3: 2) Liposomes with PE: CH: PS (3: 1: 1), PE: CH: PI: PS (3: 1: 0.5: 0.5) and PE: PC: PI: PS (1.5: 1.5: 1: 1) Was prepared as described above except that all liposomes encapsulated 1 mM NB-DNJ in 1 × PBS. Cell viability after 5 days incubation with liposomes encapsulating 1 mM NB-DNJ was determined as described above.

12.2. PBMC viability after treatment with liposomes encapsulating 1 mM NB-DNJ:

16 shows the viability of PBMCs after 5 days incubation with different liposome formulations encapsulating 1 mM NB-DNJ. Final lipid concentrations in the medium ranged from 0 to 500 μM. The results represent the mean value of three samples from three independent experiments.

The results in FIG. 16 demonstrate that encapsulation of 1 mM NB-DNJ inside liposomes does not have additional cytotoxic activity. Surprisingly, encapsulation of NB-DNJ inside certain liposomes appeared to increase cell proliferation by 160% compared to mock-treated controls.

13. Secretion of HCV from Huh7.5 cells treated with ER liposomes

The purpose of these experiments was to monitor changes in the level of HCV-1 secretion from HCV-infected Huh7.5 cells treated with different liposome compositions.

13.1. Methods for Single Cyclic HCV Secretion Assay:

Assays were performed on 8 days post infection (acute) and 50 days post infection (chronic) cells. After HCV-infected Huh7.5 cells were grown to 75% confluence in 6 well plates, the medium was replaced with 2 ml total volume of complete DMEM + 50 μM liposomes per well and 37 ° C./5% for 72 hours. Incubate at CO ₂ . All assays were performed with three samples. Virus secretion assays were performed by quantitative PCR on viral RNA extracted from 500 μl of supernatant using the Qiagen QIAamp viral RNA purification kit according to the manufacturer's protocol. The isolated RNA was first converted to cDNA using a reverse transcriptase reaction, followed by quantification of viral RNA secreted by real-time PCR using a SyBr green mix and primers oriented against HCV cDNA.

13.2. Results from single circulating secretion assay:

17 shows secretion of HCV from acutely and chronically-infected Huh7.5 cells after treatment with liposomes for 5 days. All liposomes encapsulated 1 × PBS solution, which was added to the cell culture medium at a final lipid concentration of 50 μM. HCV secretion was calculated according to the quantification of RNA in the supernatant of treated and untreated Huh7.5 cells by quantitative PCR. Results are expressed as percentage of HCV RNA secretion compared to the untreated control and represent the average of three samples.

14. Infectivity of HCV Secreted from Huh7.5 Cells Treated with ER Liposomes

The purpose of these experiments was to monitor the infectivity of HCV virions secreted from HCV-infected Huh7.5 cells treated with different liposome compositions.

14.1. Methods for Single-Circulating HCV Infectious Assays:

Supernatants containing HCV virions secreted from liposome-treated cells as described in the section above were used to determine the infectivity of HCV virions secreted from Huh7.5 cells treated with liposomes. Uninfected Huh7.5 cells were grown to 75% confluence in 48-well plates, and then the medium was replaced with 200 μl of supernatant containing HCV secreted from liposome-treated cells. The supernatant was left to infect uninfected Huh7.5 cells for 1 hour, then the cells were washed twice with 1 × PBS and then incubated at 37 ° C./5% CO ₂ for 2 days in 500 μl complete DMEM. After 2-day incubation, cells were washed twice with 1 × PBS, fixed in methanol / acetone (1: 1, vol / vol) for 10 minutes and washed twice in 1 × PBS / 0.1% Tween-20. The cells are then incubated for 1 hour in 1 × PBS / 0.1% Tween-20 containing 4 μg / ml anti-HCV core antibody, washed twice in 1 × PBS / 0.1% Tween-20, and 4 μg / ml FITC- Incubate for 1 hour in 1 × PBS / 0.1% Tween-20 containing labeled secondary antibody, wash twice more and stain with DAPI. Fluorescence images were taken using a Nikon Eclipse TE2000-U microscope as described above. The percentage of infected cells was calculated by counting the total number of cells infected with HCV (detected by anti-HCV antibodies) divided by the total number of cells in the assay (detected by DAPI staining).

14.2. Results from single-circulating HCV infectious assays:

18 shows the infectivity of HCV virions secreted from liposome-treated HCV-infected Huh7.5 cells, both acutely and chronically infected. Secreted virus particles are used to infect uninfected Huh7.5 cells, infect the cells by removing the supernatant and leaving the cells untreated for 2 days and then measuring the presence of HCV core protein in the uninfected cells. The ability was determined. Results are expressed as percentage of HCV infectivity compared to untreated control and represent the mean of three samples.

The results from HCV-infected Huh7.5 cells treated by the selection of ER liposomes and pH-sensitive liposomes (PE: CH) show that all liposomes increase the secretion of viral particles, but the infectious particles of the secreted particles are not treated. It is suggested that it is considerably reduced in comparison with.

15. ER liposomes reduce the formation of LD in Huh7.5 cells.

Huh7.5 cells were incubated overnight in the presence of ER liposomes to monitor the effect of the cells on LD. LD was visualized by confocal microscopy in liposome-treated cells.

15.1. To visualize LD in Huh7.5 cells:

ER liposome PE: PC: PI: PS (1.5: 1.7: 1.5: 0.3) was prepared as described above. Huh7.5 cells were allowed to adhere overnight on a number 1.5 glass cover slide, then the medium was exchanged and replaced with fresh medium containing liposomes added at a final lipid concentration of 50 μM. After 16 hours incubation at 37 ° C./5% CO ₂ , remove the medium containing liposomes, wash cells with 1 × PBS, fix for 15 minutes in 4% paraformaldehyde diluted in 1 × PBS, wash twice in 1 × PBS It was. Cells were then incubated with 1 × PBS containing 20 μg / ml BODIPY 493/503 for 10 minutes and washed twice in 1 × PBS. BODIPY 493/503 was suitable for detailed analysis of the microenvironment around LD. Cells were stained with DAPI and then mounted on microscope slides. Confocal images were taken using a Carl Zeiss LSM microscope and image analysis was performed using LSM software v5.10.

15.2. Results from visualization of LD inside Huh7.5 cells after 16 hours treatment with ER liposomes:

19 shows untreated Huh7.5 cells (left panel) and PE: PC: PI: PS liposome-treated Huh7.5 cells probed with BODIPY 493/503 (green) to visualize LD after 16 hour incubation (FIG. Experimental results for the right panel). PE: PC: PI: PS liposomes were added to the cell culture medium at a final lipid concentration of 50 μM. DAPI (blue) was used as a nuclear stain and the image intensity was normalized.

The results suggested that treatment of Huh7.5 cells with PE: PI: PS: PC liposomes reduced the formation of LD.

16. ER liposomes co-localize with LD in Huh7.5 cells.

Since PE: PC: PI: PS liposomes have been shown to interfere with LD formation in Huh7.5 cells, the following experiments were performed to determine whether these liposomes interact directly with the LD of the cells. After Rh-PE labeled liposomes were incubated with Huh7.5 cells for 2 hours, co-localization was determined by visualizing Rh-PE lipids and LDs of cells by confocal microscopy.

16.1. Methods of visualization of intracellular co-localization of LD and liposomes:

ER liposome PE: PC: PI: PS (1.5: 1.7: 1.5: 0.3) was prepared as described above and included 1% (total moles) of Rh-PE for visualization. Huh7.5 cells were allowed to adhere overnight on a number 1.5 glass cover slide, then the medium was exchanged and replaced with fresh medium containing Rh-PE labeled liposomes added at a final lipid concentration of 50 μM. After 2 hours incubation at 37 ° C./5% CO ₂ , the medium containing liposomes was removed, cells were fixed and stained with BODIPY 493/503 as described above. Cells were stained with DAPI and then mounted on microscope slides. Confocal images were taken as described above.

16.2. Co-localization of liposomes with Huh7.5 LD after 2 hours incubation:

20 shows the experimental results for Huh7.5 cells treated with PE: PC: PI: PS liposomes (red) for 2 hours and probed with LD stain (green). PE: PC: PI: PS liposomes were added to the cell culture medium at a final lipid concentration of 50 μM. DAPI (blue) was used as nuclear stain. The bottom-right panel is an integrated image. Yellow identifies the area of co-localization in the cells.

The results suggested that PE: PI: PS: PC liposomes could interact with LD in Huh7.5 cells after only 2 hours treatment.

17. Treatment of HCV-infected Huh7.5 cells with ER liposomes inhibits the association of LD and HCV core proteins.

Disruption of the interaction between the HCV core protein and the LD of the cell can result in the secretion of most non-infectious viral particles from HCV-infected cells.

The purpose of these experiments was to determine if liposome treatment reduced co-localization of HCV core protein and LD in Huh7.5 cells.

17.1. Methods of visualization of intracellular co-localization of LD and HCV core proteins:

ER liposome PE: PC: PI: PS (1.5: 1.7: 1.5: 0.3) was prepared as described above. After 8 days of infection with HCV genotype JFH1, Huh7.5 cells were allowed to adhere overnight on a number 1.5 glass cover slide, then the medium was exchanged and replaced with fresh medium containing liposomes added at a final lipid concentration of 50 μM. . After 16 hours incubation at 37 ° C./5% CO ₂ , remove the medium containing liposomes, wash cells twice with 1 × PBS, fix in methanol / acetone (1: 1, vol / vol) for 10 minutes, Wash twice in 1 × PBS / 0.1% Tween-20. Cells are then incubated in 1 × PBS / 0.1% Tween-20 containing 3 μg / ml anti-HCV core antibody for 1 hour, washed twice in 1 × PBS / 0.1% Tween-20, and 4 μg / ml Alexafluor (AlexaFluor) Incubated for 1 hour in 1 × PBS / 0.1% Tween-20 containing 550-labeled secondary antibody and washed twice more. Cells were then incubated with 1 × PBS containing 20 μg / ml BODIPY 493/503 for 10 minutes, washed twice in 1 × PBS, then DAPI stained and mounted as described above. Confocal images were taken as described above.

17.2. Co-localization of HCV core protein with Huh7.5 LD after 16 hours treatment with ER liposomes:

Figure 21A shows untreated Huh7.5 cells (left panel) and PE: PC: PI: PS liposome-treated Huh7.5 incubated for 16 hours and probed with anti-HCV core antibody (red) and LD stain (green). The experimental results for the cells (right panel) are shown. PE: PC: PI: PS liposomes were added to the cell culture medium at a final lipid concentration of 50 μM. DAPI (blue) was used as nuclear stain. The bottom-right panel was an integrated image. Yellow identifies the area of co-localization in the cells. 21B shows a close-up of an integrated image (white box) for both untreated (left) and PE: PC: PI: PS liposome-treated (right) cells. 21C is a schematic of HCV core protein / LD interactions in the presence (right) and absence (left) of PE: PC: PI: PS liposomes.

The presence of large LD / HCV core vesicles may be essential for the production of infectious viral particles. These results demonstrated that treatment of HCV-infected Huh7.5 cells with PE: PC: PI: PS liposomes could reduce the association of LD and HCV cores of cells, possibly secreting from ER liposome-treated cells. The reduced infectivity of HCV particles may be explained.

18. Reduced HCV secretion and infectivity by delivery of polyunsaturated lipids to HCV-infected Huh7.5 cells via ER liposomes

To improve the antiviral activity of ER liposomes against HCV, PE and PC lipids (currently 18: 1 monounsaturated in all experiments) will be replaced with polyunsaturated PE and PC (22: 6 and / or 20: 4). Could.

22A-22D show the chemical structures of polyunsaturated lipids incorporated into polyunsaturated ER liposomes. A. 22: 6 PE. B. 20: 4 PE. C. 22: 6 PC. D. 20: 4 pcs. To study the potential role of ER liposomes as HCV antiviral agents, JC-1-infected Huh7.5 cells were treated with various liposome compositions to monitor their effects on HCVcc secretion and infectivity. 22: 6 ER liposomes (22: 6 PE: 22: 6 PC: PI: PS, 1.5: 1.5: 1: 1) and 22: 6 PEG-ER liposomes (22: 6 multiplex containing 3% PEG-PE lipids) In addition to unsaturated ER liposomes, 20: 4 ER liposomes (20: 4 PE: 20: 4 PC: PI: PS, 1.5: 1.5: 1: 1) and 18: 1 ER liposomes (18: 1 PE: 18: 1 PC : PI: PS, 1.5: 1.5: 1: 1) was included to monitor the effect of different liposome lipid saturation on HCV replication.

18.1. Methods for monitoring HCVcc secretion and infectivity after liposome treatment:

The method was as described in Sections 13 & 14 above for acute JC-1 HCVcc infection.

18.2. HCVcc secretion during 4 days treatment with liposomes:

As shown in FIG. 23A, both 18: 1 and 20: 4 lipids resulted in increased HCVcc secretion compared to untreated control samples (218%, SD = 34.4% and 159%, SD = 21.6%, respectively). ). Only 22: 6 ER liposomes were found to significantly reduce HCV secretion by 27% (SD = 11.3%) at a concentration of 50 μM; Similar reductions were observed in 50 μM 22: 6 PEG-ER liposome-treatment (23%, SD = 6.6%). To determine the infectivity of secreted viral particles, supernatants from liposome-treated HCVccs were used to infect uninfected Huh7.5 cells and the number of infected cells after 48 hours infection was quantified. Figure 23B shows a significant decrease in HCV infectivity in all liposome treatments, even 18: 1 and 20: 4 ER liposome treatments resulted in increased virus secretion. Treatment with 50 μM 22: 6 ER liposomes reduced HCV infectivity by 91% (SD = 2.2%). Even the lowest concentration of 22: 6 ER liposomes tested, 1 μM, reduced infectivity by 52% (SD = 5.3%), indicating that 22: 6 polyunsaturated (pu) ER liposomes are potent inhibitors of viral infectivity. Suggested.

23A shows JC-1 HCVcc secretion from infected Huh7.5 cells (MOI = 0.5) during 4 day incubation in the presence of various ER liposome formulations quantified from 500 μl of supernatant of cells. Secretion was measured by quantification of JC-1 HCVcc RNA in the supernatant by quantitative PCR.

Figure 23B shows infectivity of secreted JC-1 HCVcc from liposome-treated JC-1-infected Huh7.5 cells. Infect uninfected Huh7.5 cells for 1 hour, then incubate for 48 hours, fix cells at this point, stain with anti-HCV core antibody to quantify the number of infected cells, and visualize all cells Infectivity of secreted HCVcc was determined by staining with DAPI.

The data in FIGS. 23A-23B may suggest that ER liposomes containing lipid 22: 6 can significantly reduce the infectivity of secreted HCV virions similar to the ER liposomes (18: 1 lipids) described above. ER liposomes containing 22: 6 polyunsaturated lipids are currently popular in the development of anti-HCV therapies.

* * *

While the above description refers to certain preferred embodiments, it will be understood that the invention is not limited thereto. It will be common to those skilled in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be included within the scope of the present invention.

All publications, patent applications, and patents cited herein are hereby incorporated by reference in their entirety.

Claims (106)

Administering a composition comprising a lipid particle comprising one or more PS lipids to a host in need of drug delivery
Comprising a drug delivery method. The method of claim 1, wherein the lipid particle is a liposome. The method of claim 1, wherein the lipid particles further comprise one or more of PE, CHEMS, PI, PC or SP lipids. The method of claim 3, wherein the lipid particles comprise PE lipids and the molar ratio between PE lipids and PS lipids is in the range of 0.5: 1 to 20: 1. The method of claim 4, wherein the molar ratio between PE lipids and PS lipids in the lipid particles ranges from 1: 1 to 10: 1. The method of claim 5, wherein the PE lipid comprises a DOPE lipid and a PEG-PE lipid. The method of claim 3, wherein the lipid particles comprise PE, PI and PC lipids. The method of claim 1, which is applied for the treatment or prevention of a disease or condition caused by or associated with a virus. The method of claim 8, wherein the disease or condition is a viral infection. The method of claim 8, wherein said administration results in the incorporation of one or more lipids of the lipid particles into the endoplasmic reticulum membrane of the cell infected with the virus. The method of claim 8, wherein the virus belongs to the family Flaviviridae. The method of claim 11, wherein the infection is a hepatitis C infection. The method of claim 12, wherein said administration reduces the production of lipid droplets in cells infected with hepatitis C virus. The method of claim 12, wherein said administration inhibits the interaction of lipid droplets with the core protein of hepatitis C virus. The method of claim 12, wherein said administration reduces the infectivity of the hepatitis C virus. The method of claim 8, wherein the virus belongs to the Retroviridae family. The method of claim 16, wherein the virus is an HIV-1 virus. The method of claim 1, wherein the composition further comprises one or more active agents encapsulated with lipid particles. The method of claim 18, wherein said administration results in the delivery of one or more active agents of the cell to the endoplasmic reticulum infected with the virus causing the infection. The method of claim 18, wherein the at least one active agent comprises an iminosugar. The method of claim 18, wherein the at least one active agent comprises an alpha glucosidase inhibitor. The method of claim 18, wherein the at least one active agent comprises an ion channel inhibitor. The method of claim 18, wherein the at least one active agent comprises N-substituted deoxynojirimycin. The method of claim 18, wherein the one or more active agents comprises N-butyl deoxynojirimycin. The method of claim 18, wherein the one or more active agents comprise one or more anti-HIV agents. The method of claim 18, wherein the one or more active agents comprise one or more antihepatitis agents. The method of claim 18, wherein the one or more active agents comprises one or more of an immunostimulant or immunomodulator and a nucleotide or nucleoside antiviral agent. The method of claim 1, wherein the composition further comprises an antiviral protein. The method of claim 28, wherein the antiviral protein is inserted into or conjugated with the lipid layer or bilayer of the lipid particle. The method of claim 1, wherein the composition comprises a targeting moiety conjugated to or incorporated into the lipid layer or bilayer of the lipid particle. The method of claim 30, wherein the targeting moiety comprises a gp120 / gp41 targeting moiety. The method of claim 30, wherein the targeting moiety comprises an sCD4 molecule. The method of claim 30, wherein the targeting moiety comprises a monoclonal antibody. The method of claim 30, wherein the targeting moiety comprises an E1 or E2 targeting moiety. The method of claim 1, wherein the host is a human. Administering to a host in need of treatment or prevention of an HIV infection a composition comprising a lipid particle comprising at least one of a PS lipid or a PI lipid and not containing a CHEMS lipid
A method of treating or preventing HIV infection, comprising. The method of claim 36, wherein the lipid particle is a liposome. 37. The method of claim 36, wherein the lipid particles further comprise PE lipids. The method of claim 38, wherein the PE lipid comprises one or more of DOPE lipids or PEG-PE lipids. The method of claim 36, wherein the lipid particle further comprises PC lipid. The method of claim 36, wherein the composition comprises one or more anti-HIV agents encapsulated in lipid particles. The method of claim 41, wherein the one or more anti-HIV agents comprise iminosugars. The method of claim 41, wherein the at least one anti-HIV agent comprises an alpha glucosidase inhibitor. The method of claim 41, wherein the at least one anti-HIV agent comprises N-substituted deoxynojirimycin. 45. The method of claim 44, wherein the at least one anti-HIV agent comprises N-butyl deoxynojirimycin. The method of claim 36, wherein the composition further comprises a targeting moiety conjugated to or embedded in a lipid layer or bilayer of the lipid particle. The method of claim 46, wherein the targeting moiety comprises a gp120 / gp41 targeting moiety. The method of claim 46, wherein the targeting moiety comprises an sCD4 molecule. The method of claim 46, wherein the targeting moiety comprises a monoclonal antibody. A method of drug delivery comprising administering to a subject in need of drug delivery a composition comprising a lipid particle comprising one or more polyunsaturated lipids. 51. The method of claim 50, wherein the lipid particle is a liposome. 51. The method of claim 50, wherein the lipid particle comprises at least one of polyunsaturated PE lipid or polyunsaturated PC lipid. The method of claim 52, wherein the lipid particles comprise polyunsaturated PE lipids and polyunsaturated PC lipids. The method of claim 52, wherein the lipid particles comprise polyunsaturated 22: 6 PE lipids. The method of claim 52, wherein the lipid particles comprise polyunsaturated 22: 6 PC lipids. The method of claim 52, wherein the lipid particles comprise polyunsaturated 22: 6 PC lipids and polyunsaturated 22: 6 PE lipids. The method of claim 52, which is applied for the treatment or prevention of a disease or condition caused by or associated with a virus. The method of claim 57, wherein the virus belongs to the Flaviviridae family. The method of claim 57, wherein the disease or condition is hepatitis C infection. 60. The method of claim 59, wherein said administration reduces HCV RNA replication. The method of claim 57, wherein the virus is an ER-germinated virus. The method of claim 57, wherein the virus is a glycoprotein containing virus. 51. The method of claim 50, wherein the composition further comprises one or more active agents encapsulated with lipid particles. 64. The method of claim 63, wherein the one or more active agents comprise iminosugars. The method of claim 63, wherein the one or more active agents comprise an alpha-glucosidase inhibitor. The method of claim 63, wherein the one or more active agents comprises an ion channel inhibitor. 64. The method of claim 63, wherein the one or more active agents comprises N-substituted deoxynojirimycin. 64. The method of claim 63, wherein the one or more active agents comprises N-butyl deoxynojirimycin. 64. The method of claim 63, wherein the one or more active agents comprises one or more antihepatitis agents. The method of claim 63, wherein the composition further comprises an antiviral protein. The method of claim 70, wherein the composition comprises a targeting moiety conjugated to or embedded in a lipid layer or bilayer of the lipid particle. 51. The method of claim 50, wherein the subject is a human. A composition comprising a lipid particle comprising a PS lipid. 74. The composition of claim 73, wherein the lipid particle is a liposome. The composition of claim 73, wherein the lipid particles further comprise one or more of PE, CHEMS, PI, PC, or SP lipids. 76. The composition of claim 75, wherein the lipid particles comprise PE lipids and the molar ratio between PE lipids and PS lipids is in the range of 0.5: 1 to 20: 1. The composition of claim 76, wherein the molar ratio between PE lipids and PS lipids in the lipid particles is in the range of 1: 1 to 10: 1. 76. The composition of claim 75, wherein the PE lipid comprises a DOPE lipid and a PEG-PE lipid. 77. The composition of claim 75, wherein the lipid particles comprise PE, PI, and PC lipids. 74. The composition of claim 73, wherein the molar concentration of PS lipids in the lipid particles is at least 10%. 81. The composition of claim 80, wherein the molar concentration of PS in the lipid particles is at least 20%. 74. The composition of claim 73, wherein the lipid particles further comprise PI lipids, wherein the combined molar concentration of PS lipids and PI lipids in the lipid particles is at least 10%. 83. The composition of claim 82, wherein the combined molar concentration of PS lipids and PI lipids in the lipid particles is at least 20%. A composition comprising a lipid particle comprising one or more polyunsaturated lipids. 85. The composition of claim 84, wherein the lipid particle is a liposome. 85. The composition of claim 84, wherein the lipid particles comprise one or more of polyunsaturated PE lipids or polyunsaturated PC lipids. 85. The composition of claim 84, wherein the lipid particles comprise polyunsaturated PE lipids and polyunsaturated PC lipids. 85. The composition of claim 84, wherein the lipid particles comprise polyunsaturated 22: 6 PE lipids. 85. The composition of claim 84, wherein the lipid particles comprise polyunsaturated 22: 6 PC lipids. 85. The composition of claim 84, wherein the lipid particles comprise polyunsaturated 22: 6 PC lipids and polyunsaturated 22: 6 PE lipids. 85. The composition of claim 84, wherein the molar concentration of polyunsaturated lipids in the lipid particles is at least 20%. A method comprising contacting a cell with a lipid particle comprising a) at least one of a PI or PS lipid and b) at least one labeled lipid comprising at least one label. 93. The method of claim 92, wherein the lipid particle is a liposome. 93. The method of claim 92, wherein the lipid particle further comprises at least one of PE and CHEMS lipid. 95. The method of claim 94, wherein the lipid particles comprise PE lipids and the molar ratio between PE lipids and PS lipids is in the range of 0.5: 1 to 20: 1. 97. The method of claim 95, wherein the molar ratio between PE lipid and PS lipid in the lipid particle is in the range of 1: 1 to 10: 1. 95. The method of claim 94, wherein the PE lipid comprises a DOPE lipid and a PEG-PE lipid. 93. The method of claim 92, wherein the lipid particles comprise PS, PE, PI, and PC lipids. 93. The method of claim 92, wherein the virus is an ER-germinated virus. 103. The method of claim 99, wherein the virus is an HCV virus or an HBV virus. 93. The method of claim 92, wherein the at least one labeled lipid comprises a fluorophore-lipid conjugate. 93. The method of claim 92, wherein the at least one labeled lipid comprises a biotin-lipid conjugate. 93. The method of claim 92, wherein said contact results in labeling the ER membrane of the cell with a label. 93. The method of claim 92, wherein the cell is a cell infected with ER-emerging virus and the contact results in labeling the viral particles emerging from the cell with a label. 107. The method of claim 104, further comprising purifying the labeled virus particles. 105. The method of claim 104, further comprising imaging labeled viral particles.

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